Compounds

ABSTRACT

Binding members, e.g. human antibody molecules, which bind interleukin-6 (IL-6) and neutralise its biological effects. Use of binding members for IL-6 in medical treatment e.g. for treating inflammatory diseases and tumours associated with IL-6.

This application is a continuation of U.S. application Ser. No.15/639,949, filed Jun. 30, 2017, which is a continuation of U.S.application Ser. No. 14/675,980, filed Apr. 1, 2015, which is acontinuation of U.S. application Ser. No. 13/480,690, filed May 25,2012, now U.S. Pat. No. 9,005,620, which is a continuation of Ser. No.11/948,659, filed Nov. 30, 2007, now U.S. Pat. No. 8,198,414, whichclaims priority to U.S. Provisional Application No. 60/861,704, filedNov. 30, 2006, the disclosures of which are hereby incorporated byreference in their entirety.

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy created on Mar. 20, 2019, is named43223US_CRF_sequencelisting.txt and is 108,566 bytes in size.

This invention relates to binding members, especially antibodymolecules, which inhibit biological effects of IL-6. The binding membersare useful for treatment of disorders associated with IL-6, includinginflammatory diseases and tumours.

Interleukin 6 (IL-6) is a 26 kDa pleiotropic pro-inflammatory cytokineproduced by a variety of cell types, including stimulated fibroblasts,monocytes and endothelial cells, which form the major source of IL-6 invivo. Cells such as T cells, B cells, macrophages, keratinocytes,osteoblasts and several others can produce IL-6 on stimulation. IL-6 isalso expressed from tumour cell lines and tumour cells e.g. cells fromlung carcinoma, prostate cancer, myeloma, hypernephroma and cardiacmyxoma [1, 2]. Under non-inflammatory conditions, IL-6 is secreted fromadipose tissue [3].

The regulation of IL-6 expression depends on the cell type that isproducing it. In multiple myeloma cells IL-6 appears to act in apositive feedback loop—stimulating the cells to grow as well as producemore IL-6 [4, 5]. In other cell types IL-6 appears to inhibit the growthand activation of cells and may act as a negative regulator for somepro-inflammatory cytokines.

To initiate cell signalling, IL-6 binds with low affinity to atransmembrane receptor, IL-6 receptor alpha (also referred to as IL-6Rα,IL-6Ra, IL-6R, gp80 or CD126) to form a complex “IL-6:IL-6Ra”. Thiscomplex binds to the gp130 signal receptor; IL-6Rα and gp130 togetherform a high affinity IL-6 binding site, and induce the formation of ahexamer composed of two copies each of IL-6, IL-6Ra and gp130 [6]. Thetransmembrane and cytoplasmic domains of the IL-6Ra are not required forsignal transduction, as IL-6Ra also exists as a soluble secreted form(sIL-6R or sIL-6Ra). The soluble receptor is produced either bydifferential splicing of the IL-6Ra message or by proteolytic shedding.sIL-6R is capable of forming a ligand-receptor complex with IL-6,“IL-6:sIL-6Ra”. This complex can bind gp130 on cells and therebyinitiate cell signalling in gp130 positive cells, even if those cells donot express IL-6Ra. Thus, sIL-6R has the potential to widen therepertoire of cells responsive to IL-6, and is thought to play animportant role in IL-6-mediated inflammation [7].

A crystal structure of human IL-6 ligand has been elucidated [6]. Thecrystal structure of the extracellular domain of human IL-6Ra [8], andthe hexameric structure of IL-6/IL-6R/gp130 complex [9], have also beenresolved. These structures combined with mutagenesis studies haveidentified three sites on the surface of IL-6 which are involved in thefunctional activity of the IL-6 in complex with the various receptorcomponents. Site 1 residues are involved in the interaction between IL-6and IL-6Ra. Site 2 residues are involved in the interaction between IL-6and the gp130 cytokine binding domain. The residues in Site 3 of IL-6are involved in interacting with the Ig-like domain of the second gp130in the hexameric complex. A fourth site on IL-6 has also been identifiedwhere IL-6 interacts with the second molecule of IL-6 in the hexamericIL-6/IL-6R/gp130 complex [10].

A number of anti-IL-6 ligand monoclonal antibodies have been isolated.Mapping studies have been performed which show that these bind todifferent binding sites, as described above, on the surface of humanIL-6 [11, 12, 13, 14, 15].

A number of anti-IL-6Ra monoclonal antibodies have also been generatedand their binding sites on the IL-6Ra mapped [16, 14, 15, 17].

IL-6 belongs to a family of cytokines, which includes Interleukin-11(IL-11), ciliary neurotrophic factor (CNTF), Oncostatin M (OsM),Leukaemia Inhibitory Factor (LIF), cardiotrophin-like cytokine (CLC),and Cardiotrophin 1 (CT-1). Each of the members of this family havetheir own specific receptor alpha subunits and form complexes with thecommon receptor subunit gp130. Targeted disruption of the gp130 gene isembryonically lethal [18, 19]. All members of the IL-6 family can inducethe expression of acute phase proteins from hepatocytes.

IL-6 signalling involves tyrosine phosphorylation by JAK family kinases,and subsequent activation of two major intracellular signallingcascades, the SHP2/ERK MAPK and STAT1/3 pathways, leading to geneexpression via NF-IL-6 and AP-1 [18, 20].

IL-6 shows a wide spectrum of biological functions including:haematopoiesis, induction of acute phase responses, T cell activation,stimulation of antibody secretion, host defence against infection,myeloma cell and osteoclast activation [21, 22]. For a review of theeffects of IL-6 see ref [23]. IL-6 was originally identified as a B-celldifferentiation factor generated by T cells [24] but has subsequentlybeen identified as a potent activator and growth-promoting factor ofmany cell types. It induces the final maturation of B cells intoantibody producing cells and is an essential accessory factor for T cellactivation and proliferation. Studies have shown that IL-6 is involvedin the activation of auto-reactive T lymphocytes and the proliferationand differentiation of cytotoxic T cells. IL-6 has been implicated inhaematopoiesis as a cofactor causing the activation and differentiationof haemopoietic stem cells. The effect of IL-6 on the acute phaseresponse is also well documented [25]. IL-6 induces a variety of acutephase proteins including fibrinogen, alpha-anti-chymotrypsin, serumamyloid A and C-reactive protein from human hepatocytes. Acute phaseproteins control immune responses and inflammation and have effects ontissue remodelling. The serum level of IL-6 correlates well with that ofC-reactive protein in variety of pathologies suggesting a causal role ofIL-6 in the acute phase response. IL-6 has also been shown to beproduced by osteoblasts and appears to be involved in osteoclastactivation and bone resorption [26, 27, 28]. Paradoxically it has beensuggested that IL-6 not only has roles as a pro-inflammatory cytokinebut can also, in certain circumstances and cell types, dampen theeffects of other pro-inflammatory cytokines leading to a reduction ininflammation.

Because IL-6 has a variety of biological effects, the elevation of IL-6has been implicated as a key cytokine in a variety of diseaseindications. The levels of circulating IL-6 have been shown to beelevated in diseases such as rheumatoid arthritis, Castleman's disease,Juvenile idiopathic arthritis and Crohn's Disease [29]. Because of thisIL-6 has been implicated in driving the pathology in these inflammatoryindications. Furthermore, a variety of tumour types have been shown tobe stimulated by IL-6, including melanoma, renal cell carcinoma,Kaposi's sarcoma, ovarian carcinoma, lymphoma, leukaemia, multiplemyeloma, and prostate carcinoma [30]. Moreover increased circulatinglevels of IL-6 have been reported in several cancers. In some cancerindications elevated IL-6 levels has been used as prognostic indicatorsof the disease.

Because of the role of IL-6 in disease a variety of murine and chimericanti-human IL-6 monoclonal antibodies have been developed as potentialtherapies.

U.S. Pat. No. 5,856,135 describes a reshaped human antibody to IL-6,derived from a mouse monoclonal antibody “SK2”. JP-10-66582 reports achimeric antibody to IL-6, which is indicated as recognising the helix Dregion of IL-6 (site 1).

WO2004/020633 (EP1536012) describes a human scFv antibody molecule toIL-6 isolated using phage display technology. The scFv is reported tohave an affinity of 13 nM.

A murine anti-IL-6 antibody, elsilimomab (also known as B-E8) has beenused to treat patients with Multiple myeloma [31, 32] renal cellcarcinoma [33] and rheumatoid arthritis [34] and improvements in certaindiagnostic markers were seen in treated patients with all threediseases. BE-8 has also been used to treat HIV-positive patients withimmunoblastic or polymorphic large cell lymphoma [35] with relief ofsystemic symptoms (i.e. fever, sweats, cachexia) and suppression ofspontaneous growth of the lymphoma in approximately 50% of patients.

However, the rapid clearance of this antibody and possible anaphylacticreactions due to the production of human anti-mouse antibodies (HAMA) toelsilimomab has limited its use in the clinic [36].

In general, clinical use of murine monoclonal antibodies is limited, assuch antibodies frequently induce HAMA. HAMA directed against the Fcpart of the mouse immunoglobulin are often produced, resulting in rapidclearance of anti-IL-6 mAb and possible anaphylactic reaction [36]. Itis also known that the pharmacokinetics of mouse antibodies in humans isdifferent from human antibodies having shorter half lives and increasedrates of clearance.

To reduce the immunogenicity of murine antibodies in humans, chimericantibodies with mouse variable regions and human constant regions havebeen constructed. A chimeric human-mouse anti-IL-6 antibody cCLB8 (knownas CNTO 328) has been used to treat patients with multiple myeloma [5,37], with disease stabilisation seen in the majority of patients.

However, although chimeric antibodies are less immunogenic than murineMAbs, human anti-chimeric antibody (HACAs) responses have been reported[38].

Mapping studies on cCLB8 have been carried out which show it is a site Iinhibitor of IL-6 activity. Brakenhoff et al [39] demonstrated thatcCLB8 binds to IL-6 amino-terminal deletion mutants Pro46, Ser49, Glu51,Ile53, Asp54 and also binds to deletion mutants Asp62 and Met77 (albeitat reduced affinity). The same authors show that cCLB8 inhibits wildtype IL-6 but not C-terminal deletion 5 in a B9 cell proliferation assayand that cCLB8 will not bind IL-6 del C-4 which has the last 4C-terminal amino acids residues deleted. This data suggest that cCLB8binds to an epitope involving the C-terminal residues of IL-6.

Kalai et al [17] demonstrated that cCLB8 failed to recognise IL-6mutants F106E, F102E/F106E or R207E/R210E. However the antibody doesrecognise IL-6 mutants R207E and R207W. The binding of cCLB8 to mutantsR207W & R207E is approximately 50% of that compared to wild type, whichsuggests that residues F106 and R210 are involved in the cCLB8 bindingepitope and residue R207 is involved in binding but has less effect thanresidues F106 and R210. The cCLB8 binds IL-6 site-I mutants R196M,K199N/Q203L and Q203L with 100% activity compared to wild type.Brakenhoff et al [13] demonstrated that cCLB8 binds the following IL-6variants; Q182H, N183K, W185Q, W185G, W185R, T190P, Q182H/Q184P,W185R/S197N, Q187E/T190P, 1164L/L186R/M1891, which is not surprising asthe majority of these are distally separated from the IL-6 site 1residues.

The positive effect of inhibiting IL-6 signalling in cancer andinflammatory diseases has been further highlighted by the use of ahumanised anti-IL-6Ra antibody Tocilizumab (also known as hPM-1, MRA andActemra). This is a humanised version of the murine anti-IL6Ra antibodyPM-1. Treatment of patients with this antibody has proven effective in anumber of diseases including rheumatoid arthritis, Juvenile idiopathicarthritis, Crohn's disease, Myeloproliferative disorder, Castleman'sdisease and Systemic lupus erythematosus [40].

We have succeeded in isolating highly potent, high affinity bindingmembers for IL-6. Owing to their high affinity and potency, and theirperformance in functional studies as described herein, binding membersof the invention are particularly suitable for use in therapeutic and/ordiagnostic treatment of the human or animal body.

The binding members are useful for treating disorders associated withIL-6, as described in detail elsewhere herein.

A human anti-IL-6 antibody for the treatment of inflammatory diseasesand cancer provides significant advantages over existing approaches. Forexample, human antibodies do not induce HAMA or HACA responses, and havea longer in vivo half life compared with non-human or chimericantibodies.

We have also recognised that binding members for IL-6 offer significantadvantages as compared with binding members for IL-6Ra, especially interms of in vivo administration and treatment, as described elsewhereherein.

As described in more detail in the Examples, we isolated a parentantibody molecule, designated CAN022D10, with a set of CDR sequences asshown in Table 7. Through a process of optimisation we generated a panelof antibody clones: Antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19,21, 22 and 23, with CDR sequences derived from the parent CDR sequencesand having substitutions at the positions indicated in Table 7.

Thus for example it can be seen from Table 7 that Antibody 2 has aparent HCDR1 sequence in which Kabat residue 35 is replaced with Thr(SEQ ID NO: 13). Antibodies 14 and 22 contain an additional residue,i.e. an amino acid insertion, in HCDR3: Ile at Kabat residue 100D, whichis not present in the parent HCDR3 sequence SEQ ID NO: 5. Antibodies 7,8, 10, 16-19, 21 and 23 do not contain Kabat residue 95 in LCDR3,whereas the parent LCDR3 (SEQ ID NO: 10) comprises Pro at Kabat residue95. The parent HCDR3, and HCDR3 sequences of all of antibodies 2, 3, 4,5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23 have Trp at Kabat residue95 and Asp at Kabat residue 101, indicating that H95 Trp and H101 Aspmay contribute to binding and/or potency for IL-6 in binding members ofthe invention.

VH domain, VL domain and CDR sequences of the parent antibody CAN022D10,and of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and23 as described herein are shown in the appended sequence listing.

As described in more detail below, binding members according to theinvention have been shown to neutralise IL-6 with high potency.Neutralisation means inhibition of a biological activity of IL-6.Binding members of the invention may neutralise one or more activitiesof IL-6. The inhibited biological activity is typically IL-6 binding toone or more of its binding partners. For example, the inhibitedbiological activity may be binding of IL-6 to transmembrane and/orsoluble IL-6Ra. This is demonstrated in the following assays, which aredescribed briefly here and in more detail below: The TF-1 assay showsthat binding members according to the invention inhibit IL-6 binding tomembrane IL-6Ra as the TF-1 cells do not appear to produce solubleIL-6Ra. As such, the binding members of the invention therefore inhibitIL-6 binding to the membrane receptor. In the synovial fibroblast assay,binding members according to the invention inhibit IL-6 binding tosoluble IL-6Ra since sIL-6Ra needs to be added to this assay for it towork. The added IL-1beta induces production of endogenous IL-6 whichwhen inhibited by a binding member of this invention prevents VEGFproduction.

In accordance with the invention, binding of human or non-human primate,e.g. cynomolgus, IL-6 to IL-6Rα may be inhibited, e.g. a binding membermay inhibit binding of mature human IL-6 to IL-6Rα.

Inhibition in biological activity may be partial or total. Bindingmembers may inhibit IL-6 biological activity by 100%, or at least 95%,at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, or at least 50% of the activity in absence of the bindingmember.

Neutralising potency of a binding member may be determined. Potency isnormally expressed as an IC₅₀ value, in nM unless otherwise stated. Infunctional assays, IC₅₀ is the concentration of a binding member thatreduces a biological response by 50% of its maximum. In ligand-bindingstudies, IC₅₀ is the concentration that reduces formation of theligand-receptor complex by 50% of the maximal specific binding level.IC₅₀ may be calculated by plotting % of maximal biological response as afunction of the log of the binding member concentration, and using asoftware program, such as Prism (GraphPad) or Origin (Origin Labs) tofit a sigmoidal function to the data to generate IC₅₀ values. Potencymay be determined or measured using one or more assays known to theskilled person and/or as described or referred to herein.

Neutralisation of IL-6 activity by a binding member in an assaydescribed herein, e.g. the TF-1 proliferation assay or other cell-basedassays described below, indicates that the binding member binds andneutralises IL-6. Other methods that may be used for determining bindingof a binding member to IL-6 include ELISA, Western blotting,immunoprecipitation, affinity chromatography and biochemical assays.

Binding members described herein were demonstrated to bind andneutralise biological effects of endogenous human IL-6, as shown in anassay of inhibition of VEGF release from human synovial fibroblasts inresponse to endogenous human IL-6, reported in Examples 1.7 and 2.7herein. In this assay, synovial fibroblasts from rheumatoid arthritispatients produce IL-6 in response to stimulation with IL-1β and solubleIL-6Rα, leading to IL-6 induced secretion of VEGF. The IL-6 produced bythe human synovial fibroblasts thus represents endogenous human IL-6.Endogenous IL-6 is the molecular target for medical treatment in humans,so neutralisation of endogenous IL-6 is an important indicator of thetherapeutic potential of the binding members. Since the assays wereconducted with synovial fibroblasts obtained from rheumatoid arthritispatients, the results are particularly relevant to use of the bindingmembers for treating rheumatoid arthritis. Neutralising potency ofoptimised antibody molecules tested in the VEGF release assay surpassedthat of the known anti IL-6 antibody CNTO-328.

A binding member according to the invention may have an IC₅₀ of lessthan 50 nM, e.g. less than 5 nM, e.g. less than 1 nM in an assay ofinhibition of VEGF release from human synovial fibroblasts stimulatedwith 0.6 pM human IL-1β and 2.4 nM soluble human IL-6Rα.

Endogenous IL-6 is known to be a mixture of glycosylated andunglycosylated forms. Binding of a binding member of the invention toendogenous IL-6 has been demonstrated in the synovial fibroblast assaysince this assay utilises IL-6 from human synovial fibroblasts i.e.endogenous IL-6.

A binding member of the invention may inhibit IL-6 induced proliferationof TF-1 cells. TF-1 is a human premyeloid cell line established from apatient with erythroleukaemia (Kitamura et al 1989). The TF-1 cell linerequires the presence of a growth factor for survival and proliferation.The individual growth factors TF-1 cells can respond to include IL-6,GM-CSF and Oncostatin M. A binding member of the invention may have anIC₅₀ of less than 100 nM, e.g. less than 20 nM, 10 nM or 1 nM, e.g. lessthan 100 pM, 70 pM, 50 pM, 40 pM, 30 pM, 20 pM or 10 pM, in an assay forinhibition of proliferation of TF-1 cells in response to 20 pM humanIL-6. As described herein (see Example 1.5), a parent IgG “CAN022D10”was shown to have an IC50 in the TF-1 proliferation assay of about 93nM, and we subsequently generated optimised variants of CAN022D10 havingsubstantially increased potency (IC₅₀ generally less than 100 pM), asshown in Examples 2.2, 2.5 and 2.6 (Tables 3, 4 and 5, respectively).Notably, IC₅₀ values for some of the optimised clones were measured tobe low as 5 pM or less, for example the germlined IgG Antibody 7,Antibody 17 and Antibody 18, representing extremely high neutralisingpotency of these antibodies.

A binding member of the invention may inhibit IL-6 induced proliferationof B9 cells. B9 cells are a sub-clone of the murine B-cell hybridomacell line, B 13.29, selected on the basis of their specific response toIL-6. B9 cells require IL-6 for survival and proliferation and respondto very low concentrations of IL-6. As such, proliferation of thesecells in the presence of an IL-6 antibody can be assessed and theaffinity of the antibody determined. Example 2.10 herein shows thatAntibody 18 inhibited B9 cell proliferation in response to IL-6, andshowed high affinity in this assay.

Auto-antibody production in rheumatoid arthritis is mostly of the IgMclass. SKW6.4 is a clonal IgM secreting human lymphoblastoid B cellline. Upon stimulation with IL-6 these cells secrete IgM, thus thisassay was perceived to be relevant to rheumatoid arthritis. SKW6.4 cellsmay be used in an assay to determine potency of binding members forneutralising IL-6, by determining inhibition of IgM secretion inresponse to IL-6. A binding member of the invention may have an IC50 ofless than 10 pM, e.g. less than 5 pM, in an SKW6.4 cell assay ofinhibition of IgM secretion in response to 100 pM human IL-6. Antibody18 was shown to neutralise effects of IL-6 in this assay—see Example2.11 (Table 9).

The invention provides high affinity binding members for human IL-6.High affinity for IL-6 from cynomolgus monkey was also demonstrated. Abinding member of the invention may bind human IL-6 and/or cynomolgusIL-6 with a K_(D) of not more than 1 nM, e.g. not more than 100 pM, 50pM, 30 pM or 10 pM. The K_(D) may be determined by surface plasmonresonance, e.g. BIAcore®. BIAcore® measurements of affinity aredescribed herein in Example 2.9. Remarkably, the affinity of Antibodies7 and 18 was found to be beyond the limit measurable using the BIAcore®instrument, indicating a K_(D) value below 10 pM.

As described elsewhere herein, surface plasmon resonance involvespassing an analyte in fluid phase over a ligand attached to a support,and determining binding between analyte and ligand. Surface plasmonresonance may for example be performed whereby IL-6 is passed in fluidphase over a binding member attached to a support. Surface plasmonresonance data may be fitted to a monovalent analyte data model. Anaffinity constant Kd may be calculated from the ratio of rate constantskd/ka as determined by surface plasmon resonance using a monovalentanalyte data model.

Affinity of a binding member for IL-16 may alternatively be calculatedby Schild analysis, e.g. based on an assay of inhibition of TF-1 cellproliferation in response to varied concentrations of human IL-6. Abinding member of the invention may have an affinity of less than 10 pM,e.g. less than 1 pM, as calculated by Schild analysis. As reported inExample 2.10 herein, the affinity of Antibody 18 for human IL-6 wascalculated as 0.4 pM using Schild analysis.

A binding member of the invention may optionally not cross-react withone or more, or all, of the following: leukaemia inhibitory factor(LIF), ciliary neurotrophic factor (CNTF), IL-11 or oncostatin M.

A binding member of the invention may optionally not cross-react withrat IL-6, mouse IL-6 and/or dog IL-6.

Cross-reactivity of binding members for binding other proteins ornon-human IL-6 may be tested for example in a time resolved fluorescenceassay for inhibition of human IL-6 binding to the binding memberimmobilised on a support, such as the DELFIA® epitope competition assayas described in Example 1.6. For example, any or all of LIF, CNTF,IL-11, oncostatin M, rat IL-6 and mouse IL-6 may show no inhibition,less than 50% inhibition, or may have an IC50 greater than 0.5 mM orgreater than 1 mM in the time resolved fluorescence assay for inhibitionof labelled human IL-6 binding to the binding member immobilised on asupport. For example, any or all of LIF, CNTF, IL-11, oncostatin M, ratIL-6 and mouse IL-6 may show no inhibition or may have an IC₅₀ at least10- or 100-fold greater than that of unlabelled human IL-6 in the timeresolved fluorescence assay for testing cross-reactivity. In this assay,labelled wild type mature human IL-6 is used at a final concentration ofthe Kd of its interaction with the binding member.

A binding member of the invention may cross-react with cynomolgus IL-6.Cross-reactivity may be determined as inhibition of labelled human IL-6binding to the binding member immobilised on a support, in the timeresolved fluorescence assay described above. For example, cynomolgusIL-6 may have an IC₅₀ of less than 5 nM, e.g. less than 2.5 nM, e.g.about 1 nM, in this time resolved fluorescence assay. Cynomolgus IL-6may have an IC₅₀ less than 10-fold different, e.g. less than 5-folddifferent, from the IC₅₀ of unlabelled human IL-6 in this assay.

A detailed protocol for the time resolved fluorescence assay fordetermining cross-reactivity is provided in the Materials and Methodssection. Examples of cross-reactivity data obtained in this assay areshown in Table 2 in Example 1.6.

As reported in Example 2.8, binding members described herein showed highcross-reactivity with cynomolgus IL-6, and showed no or limitedcross-reactivity with rat, mouse or dog IL-6.

The cross-reactivity data indicate that the binding members describedherein bind an epitope on IL-6 that is conserved between the human andcynomolgus IL-6 sequences, and is different in the mouse, rat and dogIL-6 sequence compared with the human sequence.

The binding members described herein are believed to bind the “site 1”region of IL-6, which is the region that interacts with IL-6Rα. Bindingmembers of the invention may thus competitively inhibit IL-6 binding toIL-6Rα, thereby neutralising biological effects of IL-6 that aremediated through 6Rα.

We investigated the ability of one of the antibodies described herein,Antibody 18, to bind mutant human IL-6, in which mutations wereengineered in site 1 residues. As described in Example 3, we identifiedmutations in human IL-6 that resulted in reduced binding by Antibody 18,indicating that the mutated residues were involved in recognition byAntibody 18 and may form part of the epitope on IL-6 bound by thisantibody.

For example, in a time resolved fluorescence assay for inhibition oflabelled wild type human IL-6 binding to Antibody 18 immobilised on asupport, no inhibition was observed for Arg207Glu mutant human IL-6 (SEQID NO: 177), indicating that Antibody 18 binds human IL-6 at residueArg207.

Since Antibody 18 and Antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 19,21, 22 and 23 were all derived from a parent antibody CAN22C10, and allhave structurally related CDRs, all these antibody molecules areexpected to bind the same or very similar overlapping epitope.Accordingly, the epitope mapping results obtained with Antibody 18 arealso expected to be representative for CAN22D10 the other optimisedantibodies described herein.

A binding member of the invention may bind human IL-6 at Phe102 and/orSer204. A binding member of the invention may also bind human IL-6 atArg207. Optionally a binding member may bind flanking residues orstructurally neighbouring residues in the IL-6 molecule, in addition tobinding Phe102 and/or Ser 204. By convention, residue numberingcorresponds to full length human IL-6 (SEQ ID NO: 161). However, bindingmay be determined using mature human IL-6. Binding to IL-6 residues isas determined by site directed mutagenesis, as explained below.

Mutagenesis of single amino acids and regions of proteins in order tocorrelate structure with activity is well known to one skilled in theart and has been used to define regions of proteins that bind toantibodies [41]. Binding to and/or neutralisation of mutant human IL-6may be used to assess whether a binding member binds Phe102, Ser204and/or Arg207. Absence of binding or neutralisation, or significantlyreduced binding or neutralisation, with mutant IL-6 compared withwild-type indicates that a binding member binds the mutated residue.

Binding to a residue in IL-6 may be determined using IL-6 mutated at theselected residue in a time resolved fluorescence assay of inhibition oflabelled wild type human IL-6 binding to the binding member immobilisedon a support, wherein the labelled wild type mature human IL-6 is at afinal concentration equal to the Kd of its interaction with the bindingmember. An example of this assay and competition data obtained are shownin Example 3, with results presented in Table 10. Where the mutant IL-6does not inhibit binding of labelled wild type IL-6 to the bindingmember, or where the mutant IL-6 has an IC50 greater than that ofunlabelled wild type IL-6 (e.g. more than 10-fold or 100-fold greater),this indicates that the mutated residue is bound by the binding member.Phe102Glu mutant human IL-6 (SEQ ID NO: 175), Ser204Glu mutant humanIL-6 (SEQ ID NO: 176), and/or Arg207Glu mutant human IL-6 (SEQ ID NO:177) may show no inhibition, or may have an IC₅₀ more than 100 foldgreater than the IC₅₀ of wild type human IL-6 (SEQ ID NO: 165), in atime resolved fluorescence assay for inhibition of labelled wild typehuman IL-6 binding to a binding member of the invention immobilised on asupport, wherein the labelled wild type human IL-6 is at a finalconcentration equal to the Kd of its interaction with the bindingmember.

A binding member of the invention may optionally not bind and/orneutralise mutant human IL-6 having a mutation at residue Phe102, Ser204and/or Arg207, e.g. mutation Phe102Glu, Ser204Glu, Ser204Tyr and/orArg207Glu. Examples of mutant human IL-6 sequences are SEQ ID NOS:175-177). Thus, a binding member of the invention may not inhibitbinding of one or more of these mutant IL-6 molecules to IL-6Rα.

A binding member of the invention may comprise an antibody molecule,e.g. a human antibody molecule. The binding member normally comprises anantibody VH and/or VL domain. VH and VL domains of binding members arealso provided as part of the invention. Within each of the VH and VLdomains are complementarity determining regions, (“CDRs”), and frameworkregions, (“FRs”). A VH domain comprises a set of HCDRs, and a VL domaincomprises a set of LCDRs. An antibody molecule may comprise an antibodyVH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It mayalternatively or also comprise an antibody VL domain comprising a VLCDR1, CDR2 and CDR3 and a framework.

A VH or VL domain framework comprises four framework regions, FR1, FR2,FR3 and FR4, interspersed with CDRs in the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Examples of antibody VH and VL domains and CDRs according to the presentinvention are as listed in the appended sequence listing that forms partof the present disclosure. Further CDRs are disclosed below and in Table7. All VH and VL sequences, CDR sequences, sets of CDRs and sets ofHCDRs and sets of LCDRs disclosed herein represent aspects andembodiments of the invention. As described herein, a “set of CDRs”comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1,HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3.Unless otherwise stated, a “set of CDRs” includes HCDRs and LCDRs.Typically binding members of the invention are monoclonal antibodies.

A binding member of the invention may comprise an antigen-binding sitewithin a non-antibody molecule, normally provided by one or more CDRse.g. a set of CDRs in a non-antibody protein scaffold, as discussedfurther below.

Described herein is a binding member comprising the parent set of CDRsas shown in Table 7 for parent CAN022D10, in which HCDR1 is SEQ ID NO: 3(Kabat residues 31-35), HCDR2 is SEQ ID NO: 4 (Kabat residues 50-65),HCDR3 is SEQ ID NO: 5 (Kabat residues 95-102), LCDR1 is SEQ ID NO: 8(Kabat residues 24-34), LCDR2 is SEQ ID NO: 9 (Kabat residues 50-56) andLCDR3 is SEQ ID NO: 10 (Kabat residues 89-97).

A binding member of the invention may comprise one or more CDRs asdescribed herein, e.g. a CDR3, and optionally also a CDR1 and CDR2 toform a set of CDRs. The CDR or set of CDRs may be a parent CDR or parentset of CDRs, or may be a CDR or set of CDRs of any of antibodies 2, 3,4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 or 23, or may be a variantthereof as described herein.

For example, a binding member or a VL domain according to the inventionmay comprise an LCDR3 having amino acid sequence SEQ ID NO: 120.

A binding member may comprise a set of H and/or L CDRs of the parentantibody or any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19,21, 22 or 23 with one or more amino acid mutations within the disclosedset of H and/or L CDRs. Amino acid mutations are substitutions,deletions or insertions of one amino acid. For example, there may be upto 20, e.g. up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations e.g.substitutions, within the set of H and/or L CDRs. For example, there maybe up to 6, 5, 4, 3 or 2 mutations, e.g. substitutions, in HCDR3 and/orthere may be up to 6, 5, 4, 3, or 2 mutations, e.g. substitutions, inLCDR3. HCDR3 and/or LCDR3 may optionally contain an insertion ordeletion of one amino acid as compared with the disclosed set of Hand/or LCDRs. Substitutions may for example be at the positionssubstituted in any of Antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18,19, 21, 22 or 23, as shown in Table 7. Thus, substitutions mayoptionally be at Kabat numbers selected from the following:

Kabat residue 35 in HCDR1;

Kabat residue 64 in HCDR2;

Kabat residue 96, 97, 98, 99, 100, 100A, 100B, 100C and/or 102 in HCDR3;

Kabat residue 34 in LCDR1;

Kabat residue 89, 90, 91, 92, 93, 94, 96 or 97 in LCDR3.

The amino acid mutations may comprise mutations as shown in Table 7,e.g. amino acid substitutions as indicated.

For example, a binding member or a VH domain according to the inventionmay comprise the parent HCDR1 with Kabat residue Ile 35 replaced by Thror Val.

A binding member or a VH domain according to the invention may comprisethe parent HCDR2 with Kabat residue Lys 64 replaced by Arg.

A binding member or a VH domain may comprise the parent HCDR3 with oneor more of the following mutations:

Kabat residue Ala 96 replaced by Glu;

Kabat residue Asp 97 replaced by Glu or Asn;

Kabat residue Asp 98 replaced by Gly, Glu or His;

Kabat residue His 99 replaced by Gly or Thr;

Kabat residue Tyr 100 replaced by Pro, Asn, Arg, Trp or Ala;

Kabat residue Tyr 100A replaced by Ala, Arg, Thr, Gly, Asn, Pro or Ser;

Kabat residue 100B replaced by His, Trp, Gln, Pro or Thr;

Kabat residue Ile 100C replaced by Ala, Val, His, Tyr or Leu;

Ile inserted at Kabat residue 100D;

Kabat residue Val 102 is replaced by Leu, His, Met or Ile.

Thus, a binding member or a VH domain of the invention may comprise anHCDR3 wherein Kabat residue 100D is Ile or wherein Kabat residue 100D isabsent.

A binding member or a VL domain of the invention may comprise the parentLCDR1 in which Kabat residue Ala 34 is replaced by Thr.

A binding member of a VL domain of the invention may comprise the parentLCDR3 with one or more of the following mutations:

Kabat residue Gln 89 replaced by Met or Ala;

Kabat residue Gln 90 replaced by Asn, Ser or Ala;

Kabat residue Ser 91 replaced by Asn, Gly, Ala or His;

Kabat residue Tyr 92 replaced by Trp, Ser, Lys or Phe;

Kabat residue Ser 93 replaced by Leu, Lys, Arg or Ala;

Kabat residue Thr 94 replaced by Ala, Gly or Pro;

Kabat residue Pro 95 deleted;

Kabat residue Trp 96 replaced by Gly;

Kabat residue Thr 97 replaced by Ser.

Thus, a binding member or a VL domain of the invention may comprise anLCDR3 in which Kabat residue 95 is Pro or wherein Kabat residue 95 isabsent.

The invention provides an isolated binding member for human IL-6comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3,wherein the set of CDRs has 22 or fewer amino acid alterations, e.g. upto 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, 1 alterations or no alterations, from a set of CDRs in which:

HCDR1 has amino acid sequence SEQ ID NO: 3;

HCDR2 has amino acid sequence SEQ ID NO: 4;

10 HCDR3 has amino acid sequence SEQ ID NO: 115;

LCDR1 has amino acid sequence SEQ ID NO: 8;

LCDR2 has amino acid sequence SEQ ID NO: 9; and

LCDR3 has amino acid sequence SEQ ID NO: 120.

An amino acid alteration may be a substitution, insertion or deletion.Examples of Kabat positions that may be substituted, and examples ofresidue substitutions are discussed below, and Table 7 illustrates someof the substitutions.

As shown in Table 7, the length of HCDR3 and LCDR3 varied betweendifferent optimised antibodies described herein. Relative to the parentCDRs of CAN022D10, an insertion between Kabat residues 100 to 102 (shownin Table 7 at Kabat residue 100D) was observed in some antibodies, and adeletion between Kabat residues 92 to 97 was observed in otherantibodies. The deletion at Kabat residue 95 was not observed incombination with the insertion. Thus, it may be advantageous for thelonger, 12 residue HCDR3 sequences to be combined with the longer, 9residue LCDR3 sequences, and it may be advantageous for the shorter, 11residue HCDR3 sequences to be combined with the shorter, 8 residue LCDR3sequences.

According to the Kabat numbering system, residues of LCDR3 are numberedfrom 89 to 97. LCDR3 sequences shorter than 9 residues are not envisagedby the Kabat numbering system. In the present invention, binding membersmay have an LCDR3 shorter than 9 residues, e.g. LCDR3 may be 8 residueslong, as shown in Table 7. We number the 8 residues of LCDR3 89, 90, 91,92, 93, 94, 96 and 97, respectively. In Table 7, deletion is thus shownat Kabat residue 95. However, it will be appreciated that the effect ofthe deletion is to reduce the length of the LCDR3 sequence, and that inprinciple the deletion could be considered to be made at any of residues89 to 97, e.g. any of residues 92 to 97.

In HCDR3, the Kabat numbering system accommodates variability in CDRlength by extension of the numbering system between Kabat residues 100and 101, e.g. including residue 100A for an HCDR3 of 9 residues, plus100B for an HCDR3 of 10 residues, plus 100C for an HCDR3 of 11 residues,plus 100D for an HCDR3 of 12 residues, as appropriate. In Table 7, theinsertion of an additional amino acid in HCDR3 of some of the optimisedclones relative to the parent HCDR3 is shown at Kabat residue 100D.However, it will be appreciated that in principle this insertion may beconsidered to be made at any of Kabat residues 100 to 102.

As demonstrated herein, one or more insertions or deletions may bepresent in one or more CDRs of a binding member, e.g. an HCDR3 and/orLCDR3. For example, a binding member of the invention may comprise a setof CDRs of any of Antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19,21, 22 and 23, or a variant thereof as described herein, wherein eachCDR optionally has an insertion to increase the length of the CDR by oneresidue or has a deletion of one residue to decrease the length of theCDR by one residue. Insertions and/or deletions may be made in HCDRsand/or an LCDRs, e.g. in an HCDR3 and/or in an LCDR3.

For example, a binding member may for example comprise a set of CDRshaving 20 or fewer amino acid substitutions in a set of CDRs wherein:

HCDR1 has amino acid sequence SEQ ID NO: 3;

HCDR2 has amino acid sequence SEQ ID NO: 4;

HCDR3 has amino acid sequence SEQ ID NO: 115;

LCDR1 has amino acid sequence SEQ ID NO: 8;

LCDR2 has amino acid sequence SEQ ID NO: 9; and

LCDR3 has amino acid sequence SEQ ID NO: 120;

wherein the binding member optionally has an insertion of one residue toincrease the length of the HCDR3 or a deletion of one residue todecrease the length of the HCDR3, and/or

has an insertion of one residue to increase the length of the LCDR3 or adeletion of one residue to decrease the length of the LCDR3.

A binding member of the invention may have an insertion of one residuein HCDR3 SEQ ID NO: 115 and/or an insertion of one residue in LCDR3 SEQID NO: 120.

Insertions or deletions may be made at any point in the CDRs. Forexample, in HCDR3 insertions or deletions may be of any of Kabatresidues 95-102, e.g. any of Kabat residues 100-102. For example, inLCDR3 insertions or deletions may be of any of Kabat residues 89 to 97,e.g. any of Kabat residues 92 to 97.

A binding member or VH domain of the invention may comprise an HCDR1 inwhich Kabat residue 35 is Ile, Thr or Val.

A binding member or VH domain of the invention may comprise an HCDR2 inwhich Kabat residue 64 is Lys or Arg.

A binding member or VH domain of the invention may comprise an HCDR3 inwhich Kabat residue 95 is Trp and/or Kabat residue 101 is Asp.

A binding member or VH domain of the invention may comprise an HCDR3wherein:

Kabat residue 96 is Ala or Glu;

Kabat residue 97 is Asp, Glu or Asn;

Kabat residue 98 is Asp, Gly, Glu or His;

Kabat residue 99 is His, Gly or Thr;

Kabat residue 100 is Pro, Tyr, Asn, Arg, Trp or Ala;

Kabat residue 100A is Pro, Tyr, Ala, Arg, Thr, Gly, Asn, Pro or Ser;

Kabat residue 100B is Trp, Tyr, His, Gln, Pro or Thr;

Kabat residue 100C is Ile, Ala, Val, His, Tyr or Leu; and

Kabat residue 102 is Leu, Val, His, Met or Ile.

A binding member or VL domain of the invention may comprise an LCDR1 inwhich Kabat residue 34 is Ala or Thr.

A binding member or VL domain of the invention may comprise an LCDR3wherein:

Kabat residue 89 is Gln, Met or Ala;

Kabat residue 90 is Gln, Asn, Ser or Ala;

Kabat residue 91 is Ser, Asn, Gly, Ala or His;

Kabat residue 92 is Trp, Tyr, Ser, Lys or Phe;

Kabat residue 93 is Leu, Ser, Lys, Arg or Ala;

Kabat residue 94 is Gly, Thr, Ala or Pro;

Kabat residue 96 is Gly or Trp; and

Kabat residue 97 is Ser or Thr.

The invention provides binding members comprising an HCDR1, HCDR2 and/orHCDR3 of the parent or any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16,17, 18, 19, 21, 22 and 23, and/or an LCDR1, LCDR2 and/or LCDR3 of theparent or any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19,21, 22 and 23 e.g. a set of CDRs of the parent or any of antibodies 2,3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23 shown in Table 7.

For example, a binding member of the invention may comprise a set ofCDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein: HCDR1 is SEQID NO: 113; HCDR2 is SEQ ID NO: 114; HCDR3 is SEQ ID NO: 115; LCDR1 isSEQ ID NO: 118; LCDR2 is SEQ ID NO: 119; and LCDR3 is SEQ ID NO: 120,representing the CDRs of Antibody 18.

The binding member may comprise a set of VH CDRs of one of theseantibodies. Optionally it may also comprise a set of VL CDRs of one ofthese antibodies, and the VL CDRs may be from the same or a differentantibody as the VH CDRs.

A VH domain comprising a set of HCDRs of the parent or any of antibodies2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23, and/or a VLdomain comprising a set of LCDRs of the parent or any of antibodies 2,3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23 are also providedby the invention.

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although as discussed further below a VH or VLdomain alone may be used to bind antigen. The antibody 2 VH domain maybe paired with the antibody 2 VL domain, so that an antibodyantigen-binding site is formed comprising both the antibody 2 VH and VLdomains. Analogous embodiments are provided for the other VH and VLdomains disclosed herein. In other embodiments, the antibody 2 VH ispaired with a VL domain other than the antibody VL. Light-chainpromiscuity is well established in the art. Again, analogous embodimentsare provided by the invention for the other VH and VL domains disclosedherein.

Thus, the VH of the parent or of any of antibodies 2, 3, 4, 5, 7, 8, 10,14, 16, 17, 18, 19, 21, 22 and 23 may be paired with the VL of theparent or of any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19,21, 22 and 23.

A binding member may comprise an antibody molecule having one or moreCDRs, e.g. a set of CDRs, within an antibody framework. For example, oneor more CDRs or a set of CDRs of an antibody may be grafted into aframework (e.g. human framework) to provide an antibody molecule. Theframework regions may be of human germline gene segment sequences. Thus,the framework may be germlined, whereby one or more residues within theframework are changed to match the residues at the equivalent positionin the most similar human germline framework. The skilled person canselect a germline segment that is closest in sequence to the frameworksequence of the antibody before germlining and test the affinity oractivity of the antibodies to confirm that germlining does notsignificantly reduce antigen binding or potency in assays describedherein. Human germline gene segment sequences are known to those skilledin the art and can be accessed for example from the VBase compilation.

A binding member of the invention may be an isolated human antibodymolecule having a VH domain comprising a set of HCDRs in a humangermline framework, e.g. Vh3_DP-86_(3-66). Thus, the VH domain frameworkregions FR1, FR2 and/or FR3 may comprise framework regions of humangermline gene segment Vh3_DP-86_(3-66) and/or may be germlined bymutating framework residues to match the framework residues of thishuman germline gene segment. FR4 may comprise a framework region ofhuman germline j segment JH2. The amino acid sequence of VH FR1 may beSEQ ID NO: 167. The amino acid sequence of VH FR2 may be SEQ ID NO: 168.The amino acid sequence of VH FR3 may be SEQ ID NO: 169. The amino acidsequence of VH FR4 may be SEQ ID NO: 170.

Normally the binding member also has a VL domain comprising a set ofLCDRs, e.g. in a human germline framework, e.g. Vkl L12. Thus, the VLdomain framework regions may comprise framework regions FR1, FR2 and/orFR3 of human germline gene segment Vkl L12 and/or may be germlined bymutating framework residues to match the framework residues of thishuman germline gene segment. FR4 may comprise a framework region ofhuman germline j segment JK2. The amino acid sequence of VL FR1 may beSEQ ID NO: 171. The amino acid sequence of VL FR2 may be SEQ ID NO: 172.The amino acid sequence of VL FR3 may be SEQ ID NO: 173. The amino acidsequence of VL FR4 may be SEQ ID NO: 174.

A germlined VL domain may or may not be germlined at the Vernier residueor residues, but is normally not.

An antibody molecule or a VH domain of the invention may comprise thefollowing set of heavy chain framework regions:

FR1 SEQ ID NO: 167;

FR2 SEQ ID NO: 168;

FR3 SEQ ID NO: 169;

FR4 SEQ ID NO: 170; [0162] or may comprise the said set of heavy chainframework regions with one, two, three, four or five amino acidalterations, e.g. substitutions.

An antibody molecule or a VL domain of the invention may comprise thefollowing set of light chain framework regions:

FR1 SEQ ID NO: 171;

FR2 SEQ ID NO: 172;

FR3 SEQ ID NO: 173;

FR4 SEQ ID NO: 174;

or may comprise the said set of light chain framework regions with one,two, three, four or five amino acid alterations, e.g. substitutions.

An amino acid alteration may be a substitution, an insertion or adeletion.

For example, an antibody molecule of the invention may comprise a set ofheavy and light chain framework regions, wherein heavy chain FR1 is SEQID NO: 167;

heavy chain FR2 is SEQ ID NO: 168;

heavy chain FR3 is SEQ ID NO: 169;

heavy chain FR4 is SEQ ID NO: 170;

light chain FR1 is SEQ ID NO: 171;

light chain FR2 is SEQ ID NO: 172;

light chain FR3 is SEQ ID NO: 173;

light chain FR4 is SEQ ID NO: 174;

or may comprise the said set of heavy and light chain framework regionswith 10 or fewer, e.g. five or fewer, amino acid alterations, e.g.substitutions. For example there may be one or two amino acidsubstitutions in the said set of heavy and light chain frameworkregions.

A non-germlined antibody molecule has the same CDRs, but differentframeworks, compared with a germlined antibody molecule. Of the antibodysequences shown herein in the appended sequence listing, sequences ofantibody nos 7, 10, 17 and 18 are germlined. Germlined antibodies 2 to5, 8, 14, 16, 19 and 21 to 23 may be produced by germlining frameworkregions of the VH and VL domain sequences shown herein for theseantibodies.

The 3′ cgt codon, and corresponding Arginine residue, shown in thenucleotide and amino acid sequences for the kappa VL domains ofAntibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23respectively were included in the expressed scFv and IgG sequences ofthese antibodies. The C terminal Arginine residue of the sequencescorresponds to Kabat residue 108. The origin of this residue and itsencoding triplet cgt is explained below.

To express the light chain of the IgG, a nucleotide sequence encodingthe antibody light chain was provided, comprising a first exon encodingthe VL domain, a second exon encoding the CL domain, and an intronseparating the first exon and the second exon. Under normalcircumstances, the intron is spliced out by cellular mRNA processingmachinery, joining the 3′ end of the first exon to the 5′ end of thesecond exon. Thus, when DNA having the said nucleotide sequence wasexpressed as RNA, the first and second exons were spliced together.Translation of the spliced RNA produces a polypeptide comprising the VLdomain and CL domain.

The choice of constant domain is significant in that for kappa lightchains the bridging amino acid is arginine, formed by the cga codon,where the first cytosine is encoded in exon 1 and the guanine andadenine are encoded in exon 2.

After splicing, for Antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19,21, 22 and 23, the Arg at Kabat residue 108 is encoded by the last base(c) of the VL domain framework 4 sequence and the first two bases (gt)of the CL domain.

The Arginine residue at Kabat residue 108 may be considered to be the Cterminal residue of the VL domain of the antibody molecule.

A binding member of the invention may be one which competes for bindingto IL-6 with any binding member that (i) binds IL-6 and (ii) comprises abinding member, VH and/or VL domain, CDR e.g. HCDR3, and/or set of CDRsdisclosed herein.

Competition between binding members may be assayed easily in vitro, forexample using ELISA and/or by tagging a specific reporter molecule toone binding member which can be detected in the presence of one or moreother untagged binding members, to enable identification of bindingmembers which bind the same epitope or an overlapping epitope. Suchmethods are readily known to one of ordinary skill in the art, and aredescribed in more detail herein (see the Detailed Description, and theepitope competition assays in the Materials and Methods section of theExamples.) Thus, a further aspect of the present invention provides abinding member comprising a human antibody antigen-binding site thatcompetes with an antibody molecule, for example an antibody moleculecomprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of theparent antibody or any of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17,18, 19, 21, 22 and 23, for binding to IL-6.

In further aspects, the invention provides an isolated nucleic acidwhich comprises a sequence encoding a binding member, VH domain and/orVL domain according to the present invention, and methods of preparing abinding member, a VH domain and/or a VL domain of the invention, whichcomprise expressing said nucleic acid under conditions to bring aboutproduction of said binding member, VH domain and/or VL domain, andrecovering it.

Another aspect of the present invention provides nucleic acid, generallyisolated, encoding a VH CDR or VL CDR sequence disclosed herein.

A further aspect provides a host cell containing or transformed withnucleic acid of the invention.

Further aspects of the present invention provide for compositionscontaining binding members of the invention, and their use in methods ofbinding, inhibiting and/or neutralising IL-6, including methods oftreatment of the human or animal body by therapy.

Binding members according to the invention may be used in a method oftreatment or diagnosis, such as a method of treatment (which may includeprophylactic treatment) of a disease or disorder in the human or animalbody (e.g. in a human patient), which comprises administering to saidpatient an effective amount of a binding member of the invention.Conditions treatable in accordance with the present invention includeany in which IL-6 plays a role, as discussed in detail elsewhere herein.

These and other aspects of the invention are described in further detailbelow.

Terminology

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B” is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

5 IL-6 and IL-6 Receptor

IL-6 is interleukin 6. IL-6 may also be referred to herein as “theantigen”.

The full length amino acid sequence of human IL-6 is SEQ ID NO: 161.This sequence is cleaved in vivo to remove an N-terminal leader peptide,to produce mature IL-6. Mature human IL-6 has amino acid sequence SEQ IDNO: 165. The mature sequence represents the in vivo circulating IL-6,which is the target antigen for therapeutic and in vivo diagnosticapplications as described herein. Accordingly, IL-6 referred to hereinis normally mature human IL-6, unless otherwise indicated by context.

IL-6 may be conjugated to a detectable label, such as HIS FLAG, e.g. foruse in assays as described herein. For example, a fusion proteincomprising IL-6 conjugated to a HIS FLAG sequence may be used. Asequence of HIS FLAG tagged human IL-6 is SEQ ID NO: 162.

IL-6 receptor a, IL-6Ra, is the receptor for interleukin 6. IL-6Ra isalso known as IL-6Ra, IL-6Ra, IL-6R and CD126. IL-6Ra exists in vivo ina transmembrane form and in a soluble form. References to IL-6Ra may betransmembrane IL-6Ra and/or soluble IL-6Ra unless otherwise indicated bycontext.

IL-6 receptor referred to herein is normally human IL-6 receptor, unlessotherwise indicated. An amino acid sequence of human soluble IL-6Ra(sIL-6Ra, sIL-6R) is SEQ ID NO: 163. An amino acid sequence of humantransmembrane IL-6Ra is SEQ ID NO: 164.

IL-6 binds IL-6Ra to form a complex, IL-6:IL-6Ra. The complex may beeither soluble (with sTL-6Ra) or membrane bound (with transmembraneIL-6Ra). When the IL-6Ra is the soluble form, the complex is designatedIL-6:sIL-6Ra. References to IL-6:IL-6Ra may include IL-6 complexed withtransmembrane IL-6Ra or with soluble IL-6Ra, unless otherwise indicatedby context.

gp130

gp130 is a receptor for the IL-6:IL-6Ra complex. Cloning andcharacterisation of gp130 is reported in Hibi et al, Cell 63:1149-1157(1990). A sequence of human gp130 is set out in SEQ ID NO: 166.

Binding Member

This describes one member of a pair of molecules that bind one another.The members of a binding pair may be naturally derived or wholly orpartially synthetically produced. One member of the pair of moleculeshas an area on its surface, or a cavity, which binds to and is thereforecomplementary to a particular spatial and polar organization of theother member of the pair of molecules. Examples of types of bindingpairs are antigen-antibody, biotin-avidin, hormone-hormone receptor,receptor-ligand, enzyme-substrate. The present invention is concernedwith antigen-antibody type reactions.

A binding member normally comprises a molecule having an antigen-bindingsite. For example, a binding member may be an antibody molecule or anon-antibody protein that comprises an antigen-binding site.

An antigen binding site may be provided by means of arrangement of CDRson non-antibody protein scaffolds, such as fibronectin or cytochrome Betc. [42, 43, 44], or by randomising or mutating amino acid residues ofa loop within a protein scaffold to confer binding specificity for adesired target. Scaffolds for engineering novel binding sites inproteins have been reviewed in detail by Nygren et al. [44]. Proteinscaffolds for antibody mimics are disclosed in WO/0034784, which isherein incorporated by reference in its entirety, in which the inventorsdescribe proteins (antibody mimics) that include a fibronectin type IIIdomain having at least one randomised loop. A suitable scaffold intowhich to graft one or more CDRs, e.g. a set of HCDRs, may be provided byany domain member of the immunoglobulin gene superfamily. The scaffoldmay be a human or non-human protein. An advantage of a non-antibodyprotein scaffold is that it may provide an antigen-binding site in ascaffold molecule that is smaller and/or easier to manufacture than atleast some antibody molecules. Small size of a binding member may conferuseful physiological properties, such as an ability to enter cells,penetrate deep into tissues or reach targets within other structures, orto bind within protein cavities of the target antigen. Use of antigenbinding sites in non-antibody protein scaffolds is reviewed in Wess,2004 [45]. Typical are proteins having a stable backbone and one or morevariable loops, in which the amino acid sequence of the loop or loops isspecifically or randomly mutated to create an antigen-binding site thatbinds the target antigen. Such proteins include the IgG-binding domainsof protein A from S. aureus, transferrin, tetranectin, fibronectin (e.g.10th fibronectin type III domain), lipocalins as well asgamma-crystalline and other Affilin™ scaffolds (Scil Proteins). Examplesof other approaches include synthetic “Microbodies” based oncyclotides—small proteins having intra-molecular disulphide bonds,Microproteins (Versabodies™, Amunix) and ankyrin repeat proteins(DARPins, Molecular Partners).

In addition to antibody sequences and/or an antigen-binding site, abinding member according to the present invention may comprise otheramino acids, e.g. forming a peptide or polypeptide, such as a foldeddomain, or to impart to the molecule another functional characteristicin addition to ability to bind antigen. Binding members of the inventionmay carry a detectable label, or may be conjugated to a toxin or atargeting moiety or enzyme (e.g. via a peptidyl bond or linker). Forexample, a binding member may comprise a catalytic site (e.g. in anenzyme domain) as well as an antigen binding site, wherein the antigenbinding site binds to the antigen and thus targets the catalytic site tothe antigen. The catalytic site may inhibit biological function of theantigen, e.g by cleavage.

Although, as noted, CDRs can be carried by non-antibody scaffolds, thestructure for carrying a CDR or a set of CDRs of the invention willgenerally be an antibody heavy or light chain sequence or substantialportion thereof in which the CDR or set of CDRs is located at a locationcorresponding to the CDR or set of CDRs of naturally occurring VH and VLantibody variable domains encoded by rearranged immunoglobulin genes.The structures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat, et al., 1987 [46], and updatesthereof. A number of academic and commercial on-line resources areavailable to query this database. For example, see ref [47] and theassociated on-line resource, currently at the web address ofhttp://www.bioinf.org.uk/abs/simkab.html.

By CDR region or CDR, it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulin as definedby Kabat et al. 1991 [48], and later editions. An antibody typicallycontains 3 heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRsis used here in order to indicate, according to the case, one of theseregions or several, or even the whole, of these regions which containthe majority of the amino acid residues responsible for the binding byaffinity of the antibody for the antigen or the epitope which itrecognizes.

Among the six short CDR sequences, the third CDR of the heavy chain(HCDR3) has a greater size variability (greater diversity essentiallydue to the mechanisms of arrangement of the genes which give rise toit). It may be as short as 2 amino acids although the longest size knownis 26. CDR length may also vary according to the length that can beaccommodated by the particular underlying framework. Functionally, HCDR3plays a role in part in the determination of the specificity of theantibody [refs. 49, 50, 51, 52, 53, 54, 55, 56].

HCDR1 may be 5 amino acids long, consisting of Kabat residues 31-35.

HCDR2 may be 17 amino acids long, consisting of Kabat residues 50-65.

HCDR3 may be 11 or 12 amino acids long, consisting of Kabat residues95-102, optionally including Kabat residue 100D.

LCDR1 may be 11 amino acids long, consisting of Kabat residues 24-34.

LCDR2 may be 7 amino acids long, consisting of Kabat residues 50-56.

LCDR3 may be 8 or 9 amino acids long, consisting of Kabat residues89-97, optionally including Kabat residue 95.

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteincomprising an antibody antigen-binding site. It must be understood herethat the invention does not relate to the antibodies in natural form,that is to say they are not in their natural environment but that theyhave been able to be isolated or obtained by purification from naturalsources, or else obtained by genetic recombination, or by chemicalsynthesis, and that they can then contain unnatural amino acids as willbe described later. Antibody fragments that comprise an antibodyantigen-binding site include, but are not limited to, molecules such asFab, Fab′, Fab′-SH, scFv, Fv, dAb and Fd. Various other antibodymolecules including one or more antibody antigen-binding sites have beenengineered, including for example Fab2, Fab3, diabodies, triabodies,tetrabodies and minibodies. Antibody molecules and methods for theirconstruction and use are described in [57].

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules that bind the target antigen. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe CDRs, of an antibody to the constant regions, or constant regionsplus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400, and a large body ofsubsequent literature. A hybridoma or other cell producing an antibodymay be subject to genetic mutation or other changes, which may or maynot alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any binding member orsubstance having an antibody antigen-binding site with the requiredspecificity and/or binding to antigen. Thus, this term covers antibodyfragments and derivatives, including any polypeptide comprising anantibody antigen-binding site, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an antibody antigen-bindingsite, or equivalent, fused to another polypeptide (e.g. derived fromanother species or belonging to another antibody class or subclass) aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023, and a large body ofsubsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann & Dubel [58].Phage display, another established technique for generating bindingmembers has been described in detail in many publications, such asKontermann & Dubel [58] and WO92/01047 (discussed further below), and USpatents U.S. Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657,5,871,907, 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197,6,225,447, 6,291,650, 6,492,160, 6,521,404. Transgenic mice in which themouse antibody genes are inactivated and functionally replaced withhuman antibody genes while leaving intact other components of the mouseimmune system, can be used for isolating human antibodies [59].Humanised antibodies can be produced using techniques known in the artsuch as those disclosed in for example WO91/09967, U.S. Pat. No.5,585,089, E9592106, U.S. Pat. No. 565,332 and WO93/17105. Further,WO2004/006955 describes methods for humanising antibodies, based onselecting variable region framework sequences from human antibody genesby comparing canonical CDR structure types for CDR sequences of thevariable region of a non-human antibody to canonical CDR structure typesfor corresponding CDRs from a library of human antibody sequences, e.g.germline antibody gene segments. Human antibody variable regions havingsimilar canonical CDR structure types to the non-human CDRs torm asubset of member human antibody sequences from which to select humanframework sequences. The subset members may be further ranked by aminoacid similarity between the human and the non-human CDR sequences. Inthe method of WO2004/006955, top ranking human sequences are selected toprovide the framework sequences for constructing a chimeric antibodythat functionally replaces human CDR sequences with the non-human CDRcounterparts using the selected subset member human frameworks, therebyproviding a humanized antibody of high affinity and low immunogenicitywithout need for comparing framework sequences between the non-human andhuman antibodies. Chimeric antibodies made according to the method arealso disclosed.

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.[60] or Krebs et al. [61].

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment [62, 63, 64], which consists of a VH or a VL domain; (v)isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site [65, 66]; (viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; [67]). Fv, scFv ordiabody molecules may be stabilized by the incorporation of disulphidebridges linking the VH and VL domains [68]. Minibodies comprising a scFvjoined to a CH3 domain may also be made [69]. Other examples of bindingfragments are Fab′, which differs from Fab fragments by the addition ofa few residues at the carboxyl terminus of the heavy chain CH1 domain,including one or more cysteines from the antibody hinge region, andFab′-SH, which is a Fab′ fragment in which the cysteine residue(s) ofthe constant domains bear a free thiol group.

Qui et al. [70] described antibody molecules containing just two CDRslinked by a framework region. CDR3 from the VH or VL domain was linkedto the CDR1 or CDR2 loop of the other domain. Linkage was through the Cterminus of the selected CDR1 or CDR2 to the N terminus of the CDR3, viaa FR region. Qui et al. selected the FR region having the fewesthydrophobic patches. The best combination for the antibody tested wasfound to be VL CDR1 linked by VH FR2 to VH CDR3 (VHCDR1-VHFR2-VLCDR3).At a molecular weight of around 3 kDa, these antibody molecules offeradvantages in terms of improved tissue penetration as compared with fullimmunoglobulins (approx. 150 kDa) or scFv (approx. 28 kDa).

Antibody fragments of the invention can be obtained starting from aparent antibody molecule or any of the antibody molecules 2, 3, 4, 5, 7,8, 10, 14, 16, 17, 18, 19, 21, 22 and 23, by methods such as digestionby enzymes e.g. pepsin or papain and/or by cleavage of the disulfidebridges by chemical reduction. In another manner, the antibody fragmentscomprised in the present invention can be obtained by techniques ofgenetic recombination likewise well known to the person skilled in theart or else by peptide synthesis by means of, for example, automaticpeptide synthesizers, such as those supplied by the company AppliedBiosystems, etc., or by nucleic acid synthesis and expression.

Functional antibody fragments according to the present invention includeany functional fragment whose half-life is increased by a chemicalmodification, especially by PEGylation, or by incorporation in aliposome.

A dAb (domain antibody) is a small monomeric antigen-binding fragment ofan antibody, namely the variable region of an antibody heavy or lightchain [64]. VH dAbs occur naturally in camelids (e.g. camel, llama) andmay be produced by immunizing a camelid with a target antigen, isolatingantigen-specific B cells and directly cloning dAb genes from individualB cells. dAbs are also producible in cell culture. Their small size,good solubility and temperature stability makes them particularlyphysiologically useful and suitable for selection and affinitymaturation. Camelid VH dAbs are being developed for therapeutic useunder the name “Nanobodies™”. A binding member of the present inventionmay be a dAb comprising a VH or VL domain substantially as set outherein, or a VH or VL domain comprising a set of CDRs substantially asset out herein.

Bispecific or bifunctional antibodies form a second generation ofmonoclonal antibodies in which two different variable regions arecombined in the same molecule [71]. Their use has been demonstrated bothin the diagnostic field and in the therapy field from their capacity torecruit new effector functions or to target several molecules on thesurface of tumour cells. Where bispecific antibodies are to be used,these may be conventional bispecific antibodies, which can bemanufactured in a variety of ways [72], e.g. prepared chemically or fromhybrid hybridomas, or may be any of the bispecific antibody fragmentsmentioned above. These antibodies can be obtained by chemical methods[73, 74] or somatic methods [75, 76] but likewise and preferentially bygenetic engineering techniques which allow the heterodimerization to beforced and thus facilitate the process of purification of the antibodysought [77]. Examples of bispecific antibodies include those of theBiTE™ technology in which the binding domains of two antibodies withdifferent specificity can be used and directly linked via short flexiblepeptides. This combines two antibodies on a short single polypeptidechain. Diabodies and scFv can be constructed without an Fc region, usingonly variable domains, potentially reducing the effects ofanti-idiotypic reaction.

Bispecific antibodies can be constructed as entire IgG, as bispecificFab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies. Bispecific diabodies, as opposed tobispecific whole antibodies, may also be particularly useful becausethey can be readily constructed and expressed in E. coli. Diabodies (andmany other polypeptides, such as antibody fragments) of appropriatebinding specificities can be readily selected using phage display(WO94/13804) from libraries. If one arm of the diabody is to be keptconstant, for instance, with a specificity directed against IL-6, then alibrary can be made where the other arm is varied and an antibody ofappropriate specificity selected. Bispecific whole antibodies may bemade by alternative engineering methods as described in Ridgeway et al.,1996 [78].

Various methods are available in the art for obtaining antibodiesagainst IL-6. The antibodies may be monoclonal antibodies, especially ofhuman, murine, chimeric or humanized origin, which can be obtainedaccording to the standard methods well known to the person skilled inthe art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” [79] or to the technique of preparation from hybridomasdescribed by Kohler and Milstein [80].

Monoclonal antibodies can be obtained, for example, from the B cells ofan animal immunized against IL-6, or one of its fragments containing theepitope recognized by said monoclonal antibodies. Suitable fragments andpeptides or polypeptides comprising them are described herein, and maybe used to immunise animals to generate antibodies against IL-6. SaidIL-6, or one of its fragments, can especially be produced according tothe usual working methods, by genetic recombination starting with anucleic acid sequence contained in the cDNA sequence coding for IL-6 orfragment thereof, by peptide synthesis starting from a sequence of aminoacids comprised in the peptide sequence of the IL-6 and/or fragmentthereof.

The monoclonal antibodies can, for example, be purified on an affinitycolumn on which IL-6 or one of its fragments containing the epitoperecognized by said monoclonal antibodies, has previously beenimmobilized. More particularly, the monoclonal antibodies can bepurified by chromatography on protein A and/or G, followed or notfollowed by ion-exchange chromatography aimed at eliminating theresidual protein contaminants as well as the DNA and the LPS, in itself,followed or not followed by exclusion chromatography on Sepharose gel inorder to eliminate the potential aggregates due to the presence ofdimers or of other multimers. In one embodiment, the whole of thesetechniques can be used simultaneously or successively.

Antigen-Binding Site

This describes the part of a molecule that binds to and is complementaryto all or part of the target antigen. In an antibody molecule it isreferred to as the antibody antigen-binding site, and comprises the partof the antibody that binds to and is complementary to all or part of thetarget antigen. Where an antigen is large, an antibody may only bind toa particular part of the antigen, which part is termed an epitope. Anantibody antigen-binding site may be provided by one or more antibodyvariable domains. An antibody antigen-binding site may comprise anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

WO2006/072620 describes engineering of antigen binding sites instructural (non-CDR) loops extending between beta strands ofimmunoglobulin domains. An antigen binding site may be engineered in aregion of an antibody molecule separate from the natural location of theCDRs, e.g. in a framework region of a VH or VL domain, or in an antibodyconstant domain e.g. CH1 and/or CH3. An antigen binding site engineeredin a structural region may be additional to, or instead of, an antigenbinding site formed by sets of CDRs of a VH and VL domain. Wheremultiple antigen binding sites are present in an antibody molecule, theymay bind the same antigen (IL-6), thereby increasing valency of thebinding member. Alternatively, multiple antigen binding sites may binddifferent antigens (IL-6 and one or more another antigen), and this maybe used to add effector functions, prolong half-life or improve in vivodelivery of the antibody molecule.

Isolated

This refers to the state in which binding members of the invention, ornucleic acid encoding such binding members, will generally be inaccordance with the present invention. Thus, binding members, VH and/orVL domains, and encoding nucleic acid molecules and vectors according tothe present invention may be provided isolated and/or purified, e.g.from their natural environment, in substantially pure or homogeneousform, or, in the case of nucleic acid, free or substantially free ofnucleic acid or genes of origin other than the sequence encoding apolypeptide with the required function. Isolated members and isolatednucleic acid will be free or substantially free of material with whichthey are naturally associated, such as other polypeptides or nucleicacids with which they are found in their natural environment, or theenvironment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practised in vitro or invivo. Members and nucleic acid may be formulated with diluents oradjuvants and still for practical purposes be isolated—for example themembers will normally be mixed with gelatin or other carriers if used tocoat microtitre plates for use in immunoassays, or will be mixed withpharmaceutically acceptable carriers or diluents when used in diagnosisor therapy. Binding members may be glycosylated, either naturally or bysystems of heterologous eukaryotic cells (e.g. CHO or NSO (ECACC85110503) cells, or they may be (for example if produced by expressionin a prokaryotic cell) unglycosylated.

Heterogeneous preparations comprising anti-IL-6 antibody molecules alsoform part of the invention. For example, such preparations may bemixtures of antibodies with full-length heavy chains and heavy chainslacking the C-terminal lysine, with various degrees of glycosylationand/or with derivatized amino acids, such as cyclisation of anN-terminal glutamic acid to form a pyroglutamic acid residue.

As used herein, the phrase “substantially as set out” refers to thecharacteristic(s) of the relevant CDRs of the VH or VL domain of bindingmembers described herein will be either identical or highly similar tothe specified regions of which the sequence is set out herein. Asdescribed herein, the phrase “highly similar” with respect to specifiedregion(s) of one or more variable domains, it is contemplated that from1 to about 5, e.g. from 1 to 4, including 1 to 3, or 1 or 2, or 3 or 4,amino acid substitutions may be made in the CDR and/or VH or VL domain.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1. This FIGURE shows the effect of administration of an anti-IL-6antibody (Antibody 18) on human recombinant IL-6 induced haptoglobinincrease in the mouse in vivo.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, a binding member in accordance with the presentinvention modulates and may neutralise a biological activity of IL-6. Asdescribed herein, IL-6-binding members of the present invention may beoptimised for neutralizing potency. Generally, potency optimisationinvolves mutating the sequence of a selected binding member (normallythe variable domain sequence of an antibody) to generate a library ofbinding members, which are then assayed for potency and the more potentbinding members are selected. Thus selected “potency-optimised” bindingmembers tend to have a higher potency than the binding member from whichthe library was generated. Nevertheless, high potency binding membersmay also be obtained without optimisation, for example a high potencybinding member may be obtained directly from an initial screen e.g. abiochemical neutralization assay. A “potency optimized” binding memberrefers to a binding member with an optimized potency of neutralizationof a particular activity or downstream function of IL-6. Assays andpotencies are described in more detail elsewhere herein. The presentinvention provides both potency-optimized and non-optimized bindingmembers, as well as methods for potency optimization from a selectedbinding member. The present invention thus allows the skilled person togenerate binding members having high potency.

In a further aspect, the present invention provides a method ofobtaining one or more binding members able to bind the antigen, themethod including bringing into contact a library of binding membersaccording to the invention and said antigen, and selecting one or morebinding members of the library able to bind said antigen.

The library may be displayed on particles or molecular complexes, e.g.replicable genetic packages, such as yeast, bacterial or bacteriophage(e.g. T7) particles, viruses, cells or covalent, ribosomal or other invitro display systems, each particle or molecular complex containingnucleic acid encoding the antibody VH variable domain displayed on it,and optionally also a displayed VL domain if present. Phage display isdescribed in WO92/01047 and e.g. US patents U.S. Pat. Nos. 5,969,108,5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793,5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and6,521,404, each of which is herein incorporated by reference in theirentirety.

Following selection of binding members able to bind the antigen anddisplayed on bacteriophage or other library particles or molecularcomplexes, nucleic acid may be taken from a bacteriophage or otherparticle or molecular complex displaying a said selected binding member.Such nucleic acid may be used in subsequent production of a bindingmember or an antibody VH or VL variable domain by expression fromnucleic acid with the sequence of nucleic acid taken from abacteriophage or other particle or molecular complex displaying a saidselected binding member.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected binding member may beprovided in isolated form, as may a binding member comprising such a VHdomain.

Ability to bind IL-6 may be further tested, also ability to compete withe.g. a parent antibody molecule or an antibody molecule 2, 3, 4, 5, 7,8, 10, 14, 16, 17, 18, 19, 21, 22 or 23 (e.g. in scFv format and/or IgGformat, e.g. IgG1) for binding to IL-6. Ability to neutralize IL-6 maybe tested, as discussed further elsewhere herein.

A binding member according to the present invention may bind IL-6 withthe affinity of a parent or other antibody molecule, e.g. scFv, or oneof antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23,e.g. IgG1, or with an affinity that is better.

A binding member according to the present invention may neutralise abiological activity of IL-6 with the potency of a parent or otherantibody molecule, one of antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17,18, 19, 21, 22 and 23 e.g. scFv, or IgG1, or with a potency that isbetter.

Binding affinity and neutralization potency of different binding memberscan be compared under appropriate conditions.

Variants of the VH and VL domains and CDRs of the present invention,including those for which amino acid sequences are set out herein, andwhich can be employed in binding members of the invention can beobtained by means of methods of sequence alteration or mutation andscreening for antigen binding members with desired characteristics.Examples of desired characteristics include but are not limited to:

-   -   Increased binding affinity for antigen relative to known        antibodies which are specific for the antigen    -   Increased neutralization of an antigen activity relative to        known antibodies which are specific for the antigen if the        activity is known    -   Specified competitive ability with a known antibody or ligand to        the antigen at a specific molar ratio    -   Ability to immunoprecipitate complex    -   Ability to bind to a specified epitope        -   Linear epitope, e.g. peptide sequence identified using            peptide-binding scan as described herein, e.g. using            peptides screened in linear and/or constrained conformation        -   Conformational epitope, formed by non-continuous residues    -   Ability to modulate a new biological activity of IL-6, or        downstream molecule.        Such methods are also provided herein.

Variants of antibody molecules disclosed herein may be produced and usedin the present invention. Following the lead of computational chemistryin applying multivariate data analysis techniques to thestructure/property-activity relationships [81] quantitativeactivity-property relationships of antibodies can be derived usingwell-known mathematical techniques, such as statistical regression,pattern recognition and classification [82, 83, 84, 85, 86, 87]. Theproperties of antibodies can be derived from empirical and theoreticalmodels (for example, analysis of likely contact residues or calculatedphysicochemical property) of antibody sequence, functional andthree-dimensional structures and these properties can be consideredsingly and in combination.

An antibody antigen-binding site composed of a VH domain and a VL domainis typically formed by six loops of polypeptide: three from the lightchain variable domain (VL) and three from the heavy chain variabledomain (VH). Analysis of antibodies of known atomic structure haselucidated relationships between the sequence and three-dimensionalstructure of antibody combining sites [88, 89]. These relationshipsimply that, except for the third region (loop) in VH domains, bindingsite loops have one of a small number of main-chain conformations:canonical structures. The canonical structure formed in a particularloop has been shown to be determined by its size and the presence ofcertain residues at key sites in both the loop and in framework regions[88, 89].

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain bindingspecificity. These predictions can be backed up by comparison of thepredictions to the output from lead optimization experiments. In astructural approach, a model can be created of the antibody molecule[90] using any freely available or commercial package, such as WAM [91].A protein visualisation and analysis software package, such as InsightII (Accelrys, Inc.) or Deep View [92] may then be used to evaluatepossible substitutions at each position in the CDR. This information maythen be used to make substitutions likely to have a minimal orbeneficial effect on activity.

The techniques required to make substitutions within amino acidsequences of CDRs, antibody VH or VL domains and binding membersgenerally are available in the art. Variant sequences may be made, withsubstitutions that may or may not be predicted to have a minimal orbeneficial effect on activity, and tested for ability to bind and/orneutralize IL-6 and/or for any other desired property.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.

Variants of VL domains of the invention, and binding members or antibodymolecules comprising them, include VL domains in which Arginine is notpresent at Kabat residue 108, e.g. where Kabat residue 108 is adifferent residue or is deleted. For example, an antibody molecule, suchas an antibody molecule lacking a constant domain, e.g. an scFv, maycomprise a VL domain having a VL domain sequence or variant thereof asdescribed herein, in which Arginine at Kabat residue 108 an amino acidresidue other than Arginine or is deleted.

A further aspect of the invention is an antibody molecule comprising aVH domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acidsequence identity with a VH domain of any of antibodies 2, 3, 4, 5, 7,8, 10, 14, 16, 17, 18, 19, 21, 22 and 23 shown in the appended sequencelisting, and/or comprising a VL domain that has at least 60, 70, 80, 85,90, 95, 98 or 99% amino acid sequence identity with a VL domain of anyof antibodies 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23shown in the appended sequence listing. Algorithms that can be used tocalculate % identity of two amino acid sequences include e.g. BLAST[93], FASTA [94], or the Smith-Waterman algorithm [95], e.g. employingdefault parameters.

Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue).

Alterations may be made in one or more framework regions and/or one ormore CDRs. The alterations normally do not result in loss of function,so a binding member comprising a thus-altered amino acid sequence mayretain an ability to bind and/or neutralize IL-6. It may retain the samequantitative binding and/or neutralizing ability as a binding member inwhich the alteration is not made, e.g. as measured in an assay describedherein. The binding member comprising a thus-altered amino acid sequencemay have an improved ability to bind and/or neutralize IL-6. Alterationmay comprise replacing one or more amino acid residue with anon-naturally occurring or non-standard amino acid, modifying one ormore amino acid residue into a non-naturally occurring or non-standardform, or inserting one or more non-naturally occurring or non-standardamino acid into the sequence. Examples of numbers and locations ofalterations in sequences of the invention are described elsewhereherein. Naturally occurring amino acids include the “standard” L-aminoacids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R,H, D, E by their standard single-letter codes. Non-standard amino acidsinclude any other residue that may be incorporated into a polypeptidebackbone or result from modification of an existing amino acid residue.Non-standard amino acids may be naturally occurring or non-naturallyoccurring. Several naturally occurring non-standard amino acids areknown in the art, such as 4-hydroxyproline, 5-hydroxylysine,3-methylhistidine, N-acetylserine, etc. [96]. Those amino acid residuesthat are derivatised at their N-alpha position will only be located atthe N-terminus of an amino-acid sequence. Normally in the presentinvention an amino acid is an L-amino acid, but it may be a D-aminoacid. Alteration may therefore comprise modifying an L-amino acid into,or replacing it with, a D-amino acid. Methylated, acetylated and/orphosphorylated forms of amino acids are also known, and amino acids inthe present invention may be subject to such modification.

Amino acid sequences in antibody domains and binding members of theinvention may comprise non-natural or non-standard amino acids describedabove. Non-standard amino acids (e.g. D-amino acids) may be incorporatedinto an amino acid sequence during synthesis, or by modification orreplacement of the “original” standard amino acids after synthesis ofthe amino acid sequence.

Use of non-standard and/or non-naturally occurring amino acids increasesstructural and functional diversity, and can thus increase the potentialfor achieving desired IL-6-binding and neutralizing properties in abinding member of the invention. Additionally, D-amino acids andanalogues have been shown to have different pharmacokinetic profilescompared with standard L-amino acids, owing to in vivo degradation ofpolypeptides having L-amino acids after administration to an animal e.g.a human, meaning that D-amino acids are advantageous for some in vivoapplications.

Novel VH or VL regions carrying CDR-derived sequences of the inventionmay be generated using random mutagenesis of one or more selected VHand/or VL genes to generate mutations within the entire variable domain.Such a technique is described by Gram et al. [97], who used error-pronePCR. In some embodiments one or two amino acid substitutions are madewithin an entire variable domain or set of CDRs.

Another method that may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al. [98]and Schier et al. [99]. All the above-described techniques are known assuch in the art and the skilled person will be able to use suchtechniques to provide binding members of the invention using routinemethodology in the art.

A further aspect of the invention provides a method for obtaining anantibody antigen-binding site for IL-6, the method comprising providingby way of addition, deletion, substitution or insertion of one or moreamino acids in the amino acid sequence of a VH domain set out herein aVH domain which is an amino acid sequence variant of the VH domain,optionally combining the VH domain thus provided with one or more VLdomains, and testing the VH domain or VH/VL combination or combinationsto identify a binding member or an antibody antigen-binding site forIL-6 and optionally with one or more desired properties, e.g. ability toneutralize IL-6 activity. Said VL domain may have an amino acid sequencewhich is substantially as set out herein. An analogous method may beemployed in which one or more sequence variants of a VL domain disclosedherein are combined with one or more VH domains.

As noted above, a CDR amino acid sequence substantially as set outherein may be carried as a CDR in a human antibody variable domain or asubstantial portion thereof. The HCDR3 sequences substantially as setout herein represent embodiments of the present invention and each ofthese may be carried as a HCDR3 in a human heavy chain variable domainor a substantial portion thereof.

Variable domains employed in the invention may be obtained or derivedfrom any germline or rearranged human variable domain, or may be asynthetic variable domain based on consensus or actual sequences ofknown human variable domains. A variable domain can be derived from anon-human antibody. A CDR sequence of the invention (e.g. CDR3) may beintroduced into a repertoire of variable domains lacking a CDR (e.g.CDR3), using recombinant DNA technology. For example, Marks et al. [100]describe methods of producing repertoires of antibody variable domainsin which consensus primers directed at or adjacent to the 5′ end of thevariable domain area are used in conjunction with consensus primers tothe third framework region of human VH genes to provide a repertoire ofVH variable domains lacking a CDR3. Marks et al. further describe howthis repertoire may be combined with a CDR3 of a particular antibody.Using analogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide binding members of the invention. Therepertoire may then be displayed in a suitable host system, such as thephage display system of WO92/01047, which is herein incorporated byreference in its entirety, or any of a subsequent large body ofliterature, including Kay, Winter & McCafferty [101], so that suitablebinding members may be selected. A repertoire may consist of fromanything from 10⁴ individual members upwards, for example at least 10⁵,at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹ or at least 10¹⁰members or more. Other suitable host systems include, but are notlimited to yeast display, bacterial display, T7 display, viral display,cell display, ribosome display and covalent display.

A method of preparing a binding member for IL-6 antigen is provided,which method comprises:

-   -   (a) providing a starting repertoire of nucleic acids encoding a        VH domain which either include a CDR3 to be replaced or lack a        CDR3 encoding region;    -   (b) combining said repertoire with a donor nucleic acid encoding        an amino acid sequence substantially as set out herein for a VH        CDR3 such that said donor nucleic acid is inserted into the CDR3        region in the repertoire, so as to provide a product repertoire        of nucleic acids encoding a VH domain;    -   (c) expressing the nucleic acids of said product repertoire;    -   (d) selecting a binding member for IL-6; and    -   (e) recovering said binding member or nucleic acid encoding it.

Again, an analogous method may be employed in which a VL CDR3 of theinvention is combined with a repertoire of nucleic acids encoding a VLdomain that either include a CDR3 to be replaced or lack a CDR3 encodingregion.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains that are then screened for a bindingmember or binding members for IL-6.

For example, one or more of the parent or antibody 2, 3, 4, 5, 7, 8, 10,14, 16, 17, 18, 19, 21, 22 or 23 HCDR1, HCDR2 and HCDR3 or the parent orantibody 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 or 23 set ofHCDRs may be employed, and/or one or more of the parent or antibody 2,3, 4, 5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 or 23 LCDR1, LCDR2 andLCDR3 or the parent or antibody 2, 3, 4, 5, 7, 8, 10, 14, 16, 17, 18,19, 21, 22 or 23 set of LCDRs may be employed.

Similarly, other VH and VL domains, sets of CDRs and sets of HCDRsand/or sets of LCDRs disclosed herein may be employed.

A substantial portion of an immunoglobulin variable domain maycompriseat least the three CDR regions, together with their interveningframework regions. The portion may also include at least about 50% ofeither or both of the first and fourth framework regions, the 50% beingthe C-terminal 50% of the first framework region and the N-terminal 50%of the fourth framework region. Additional residues at the N-terminal orC-terminal end of the substantial part of the variable domain may bethose not normally associated with naturally occurring variable domainregions. For example, construction of binding members of the presentinvention made by recombinant DNA techniques may result in theintroduction of N- or C-terminal residues encoded by linkers introducedto facilitate cloning or other manipulation steps. Other manipulationsteps include the introduction of linkers to join variable domains ofthe invention to further protein sequences including antibody constantregions, other variable domains (for example in the production ofdiabodies) or detectable/functional labels as discussed in more detailelsewhere herein.

Although in some aspects of the invention, binding members comprise apair of VH and VL domains, single binding domains based on either VH orVL domain sequences form further aspects of the invention. It is knownthat single immunoglobulin domains, especially VH domains, are capableof binding target antigens in a specific manner. For example, see thediscussion of dAbs above. In the case of either of the single bindingdomains, these domains may be used to screen for complementary domainscapable of forming a two-domain binding member able to bind IL-6. Thismay be achieved by phage display screening methods using the so-calledhierarchical dual combinatorial approach as disclosed in WO92/01047,herein incorporated by reference in its entirety, in which an individualcolony containing either an H or L chain clone is used to infect acomplete library of clones encoding the other chain (L or H) and theresulting two-chain binding member is selected in accordance with phagedisplay techniques, such as those described in that reference. Thistechnique is also disclosed in Marks et al, ibid. [100].

Binding members of the present invention may further comprise antibodyconstant regions or parts thereof, e.g. human antibody constant regionsor parts thereof. For example, a VL domain may be attached at itsC-terminal end to antibody light chain constant domains including humanCκ or Cλ, chains. Similarly, a binding member based on a VH domain maybe attached at its C-terminal end to all or part (e.g. a CH1 domain) ofan immunoglobulin heavy chain derived from any antibody isotype, e.g.IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularlyIgG1 and IgG4. IgG1 is advantageous, due to its effector function andease of manufacture. Any synthetic or other constant region variant thathas these properties and stabilizes variable regions may also be usefulin the present invention.

Binding members of the invention may be labelled with a detectable orfunctional label. Thus, a binding member or antibody molecule can bepresent in the form of an immunoconjugate so as to obtain a detectableand/or quantifiable signal. An immunoconjugates may comprise an antibodymolecule of the invention conjugated with detectable or functionallabel. A label can be any molecule that produces or can be induced toproduce a signal, including but not limited to fluorescers, radiolabels,enzymes, chemiluminescers or photosensitizers. Thus, binding may bedetected and/or measured by detecting fluorescence or luminescence,radioactivity, enzyme activity or light absorbance.

Suitable labels include, by way of illustration and not limitation,

-   -   enzymes, such as alkaline phosphatase, glucose-6-phosphate        dehydrogenase (“G6PDH”), alpha-D-galactosidase, glucose oxydase,        glucose amylase, carbonic anhydrase, acetylcholinesterase,        lysozyme, malate dehydrogenase and peroxidase e.g. horseradish        peroxidase;    -   dyes;    -   fluorescent labels or fluorescers, such as fluorescein and its        derivatives, derivatives, fluorochrome, rhodamine compounds and        derivatives, GFP, (GFP for “Green Flourescent Protein”), dansyl,        unbelliferone, phycoerythrin, phycocyanin, allophycocyanin,        o-phthaldehyde, and fluorescamine; fluorophores such as        lanthanide phthaldehyde, and fluorescamine; fluorophores such as        lanthanide cryptates and chelates e.g. Europium etc (Perkin        Elmer and Cis Biointernational),    -   chemoluminescent labels or chemiluminescers, such as isoluminol,        luminol and the dioxetanes;    -   bio-luminescent labels, such as luciferase and luciferin;    -   sensitizers;    -   coenzymes;    -   enzyme substrates;    -   radiolabels including but not limited to bromine77, carbon14,        cobalt57, fluorine8, gallium67, gallium 68, hydrogen3 (tritium),        indium 111, indium 113m, iodine123m, iodine125, iodine126,        iodine131, iodine133, mercury107, mercury203, phosphorous32,        rhenium99m, rhenium 101, rhenium 105, ruthenium95, ruthenium97,        ruthenium 103, ruthenium105, scandium47, selenium75, sulphur35,        technetium99, technetium99m, tellurium 121 m, tellurium 122m,        tellurium125m, thulium 165, thulium 167, thulium 168, yttrium199        and other radiolabels mentioned herein;    -   particles, such as latex or carbon particles; metal sol;        crystallite; liposomes; cells, etc., which may be further        labelled with a dye, catalyst or other detectable group;    -   molecules such as biotin, digoxygenin or 5-bromodeoxyuridine;    -   toxin moieties, such as for example a toxin moiety selected from        a group of Pseudomonas exotoxin (PE or a cytotoxic fragment or        mutant thereof), Diptheria toxin or a cytotoxic fragment or        mutant thereof, a botulinum toxin A, B, C, D, E or F, ricin or a        cytotoxic fragment thereof e.g. ricin A, abrin or a cytotoxic        fragment thereof, saporin or a cytotoxic fragment thereof,        pokeweed antiviral toxin or a cytotoxic fragment thereof and        bryodin 1 or a cytotoxic fragment thereof.

Suitable enzymes and coenzymes are disclosed in Litman, et al., U.S.Pat. No. 4,275,149, and Boguslaski, et al., U.S. Pat. No. 4,318,980,each of which are herein incorporated by reference in their entireties.Suitable fluorescers and chemiluminescers are disclosed in Litman, etal., U.S. Pat. No. 4,275,149, which is incorporated herein by referencein its entirety. Labels further include chemical moieties, such asbiotin that may be detected via binding to a specific cognate detectablemoiety, e.g. labelled avidin or streptavidin. Detectable labels may beattached to antibodies of the invention using conventional chemistryknown in the art.

Immunoconjugates or their functional fragments can be prepared bymethods known to the person skilled in the art. They can be coupled toenzymes or to fluorescent labels directly or by the intermediary of aspacer group or of a linking group, such as a polyaldehyde, likeglutaraldehyde, ethylenediaminetetraacetic acid (EDTA),diethylene-triaminepentaacetic acid (DPTA), or in the presence ofcoupling agents, such as those mentioned above for the therapeuticconjugates. Conjugates containing labels of fluorescein type can beprepared by reaction with an isothiocyanate.

The methods known to the person skilled in the art existing for couplingthe therapeutic radioisotopes to the antibodies either directly or via achelating agent, such as EDTA, DTPA mentioned above can be used for theradioelements which can be used in diagnosis. It is likewise possible toperform labelling with sodium125 by the chloramine T method [102] orelse with technetium99m by the technique of Crockford et al., (U.S. Pat.No. 4,424,200, herein incorporated by reference in its entirety) orattached via DTPA as described by Hnatowich (U.S. Pat. No. 4,479,930,herein incorporated by reference in its entirety).

There are numerous methods by which the label can produce a signaldetectable by external means, for example, by visual examination,electromagnetic radiation, heat, and chemical reagents. The label canalso be bound to another binding member that binds the antibody of theinvention, or to a support.

The label can directly produce a signal, and therefore, additionalcomponents are not required to produce a signal. Numerous organicmolecules, for example fluorescers, are able to absorb ultraviolet andvisible light, where the light absorption transfers energy to thesemolecules and elevates them to an excited energy state. This absorbedenergy is then dissipated by emission of light at a second wavelength.This second wavelength emission may also transfer energy to a labelledacceptor molecule, and the resultant energy dissipated from the acceptormolecule by emission of light for example fluorescence resonance energytransfer (FRET). Other labels that directly produce a signal includeradioactive isotopes and dyes.

Alternately, the label may need other components to produce a signal,and the signal producing system would then include all the componentsrequired to produce a measurable signal, which may include substrates,coenzymes, enhancers, additional enzymes, substances that react withenzymic products, catalysts, activators, cofactors, inhibitors,scavengers, metal ions, and a specific binding substance required forbinding of signal generating substances. A detailed discussion ofsuitable signal producing systems can be found in Ullman, et al. U.S.Pat. No. 5,185,243, which is herein incorporated herein by reference inits entirety. The present invention provides a method comprising causingor allowing binding of a binding member as provided herein to IL-6.

As noted, such binding may take place in vivo, e.g. followingadministration of a binding member, or nucleic acid encoding a bindingmember, or it may take place in vitro, for example in ELISA, Westernblotting, immunocytochemistry, immunoprecipitation, affinitychromatography, and biochemical or cell-based assays, such as a TF-1cell proliferation assay.

The present invention also provides for measuring levels of antigendirectly, by employing a binding member according to the invention forexample in a biosensor system. For instance, the present inventioncomprises a method of detecting and/or measuring binding to IL-6,comprising, (i) exposing said binding member to IL-6 and (ii) detectingbinding of said binding member to IL-6, wherein binding is detectedusing any method or detectable label described herein. This, and anyother binding detection method described herein, may be interpreteddirectly by the person performing the method, for instance, by visuallyobserving a detectable label. Alternatively, this method, or any otherbinding detection method described herein, may produce a report in theform of an autoradiograph, a photograph, a computer printout, a flowcytometry report, a graph, a chart, a test tube or container or wellcontaining the result, or any other visual or physical representation ofa result of the method.

The amount of binding of binding member to IL-6 may be determined.Quantitation may be related to the amount of the antigen in a testsample, which may be of diagnostic interest. Screening for IL-6 bindingand/or the quantitation thereof may be useful, for instance, inscreening patients for diseases or disorders referred to herein and/orany other disease or disorder involving aberrant IL-6 expression and/oractivity.

A diagnostic method of the invention may comprise (i) obtaining a tissueor fluid sample from a subject, (ii) exposing said tissue or fluidsample to one or more binding members of the present invention; and(iii) detecting bound IL-6 as compared with a control sample, wherein anincrease in the amount of IL-6 binding as compared with the control mayindicate an aberrant level of IL-6 expression or activity. Tissue orfluid samples to be tested include blood, serum, urine, biopsy material,tumours, or any tissue suspected of containing aberrant IL-6 levels.Subjects testing positive for aberrant IL-6 levels or activity may alsobenefit from the treatment methods disclosed later herein. Those skilledin the art are able to choose a suitable mode of determining binding ofthe binding member to an antigen according to their preference andgeneral knowledge, in light of the methods disclosed herein.

The reactivities of binding members in a sample may be determined by anyappropriate means. Radioimmunoassay (RIA) is one possibility.Radioactive labelled antigen is mixed with unlabelled antigen (the testsample) and allowed to bind to the binding member. Bound antigen isphysically separated from unbound antigen and the amount of radioactiveantigen bound to the binding member determined. The more antigen thereis in the test sample the less radioactive antigen will bind to thebinding member. A competitive binding assay may also be used withnon-radioactive antigen, using antigen or an analogue linked to areporter molecule. The reporter molecule may be a fluorochrome, phosphoror laser dye with spectrally isolated absorption or emissioncharacteristics. Suitable fluorochromes include fluorescein, rhodamine,phycoerythrin and Texas Red, and lanthanide chelates or cryptates.Suitable chromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material, such as latex beads that are colored, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes, which catalyze reactions that develop, or change colours orcause changes in electrical properties, for example. They may bemolecularly excitable, such that electronic transitions between energystates result in characteristic spectral absorptions or emissions. Theymay include chemical entities used in conjunction with biosensors.Biotin/avidin or biotin/streptavidin and alkaline phosphatase detectionsystems may be employed.

The signals generated by individual binding member-reporter conjugatesmay be used to derive quantifiable absolute or relative data of therelevant binding member binding in samples (normal and test).

A kit comprising a binding member according to any aspect or embodimentof the present invention is also provided as an aspect of the presentinvention. In the kit, the binding member may be labelled to allow itsreactivity in a sample to be determined, e.g. as described furtherbelow. Further the binding member may or may not be attached to a solidsupport. Components of a kit are generally sterile and in sealed vialsor other containers. Kits may be employed in diagnostic analysis orother methods for which binding members are useful. A kit may containinstructions for use of the components in a method, e.g. a method inaccordance with the present invention. Ancillary materials to assist inor to enable performing such a method may be included within a kit ofthe invention. The ancillary materials include a second, differentbinding member which binds to the first binding member and is conjugatedto a detectable label (e.g., a fluorescent label, radioactive isotope orenzyme). Antibody-based kits may also comprise beads for conducting animmunoprecipitation. Each component of the kits is generally in its ownsuitable container. Thus, these kits generally comprise distinctcontainers suitable for each binding member. Further, the kits maycomprise instructions for performing the assay and methods forinterpreting and analyzing the data resulting from the performance ofthe assay.

The present invention also provides the use of a binding member as abovefor measuring antigen levels in a competition assay, that is to say amethod of measuring the level of antigen in a sample by employing abinding member as provided by the present invention in a competitionassay. This may be where the physical separation of bound from unboundantigen is not required. Linking a reporter molecule to the bindingmember so that a physical or optical change occurs on binding is onepossibility. The reporter molecule may directly or indirectly generatedetectable signals, which may be quantifiable. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

In various aspects and embodiments, the present invention extends to abinding member that competes for binding to IL-6 with any binding memberdefined herein, e.g. the parent antibody or any of antibodies 2, 3, 4,5, 7, 8, 10, 14, 16, 17, 18, 19, 21, 22 and 23, e.g. in IgG1 format.Competition between binding members may be assayed easily in vitro, forexample by tagging a specific reporter molecule to one binding memberwhich can be detected in the presence of other untagged bindingmember(s), to enable identification of binding members which bind thesame epitope or an overlapping epitope. Competition may be determinedfor example using ELISA in which IL-6 is immobilized to a plate and afirst tagged or labelled binding member along with one or more otheruntagged or unlabelled binding members is added to the plate. Presenceof an untagged binding member that competes with the tagged bindingmember is observed by a decrease in the signal emitted by the taggedbinding member.

For example, the present invention includes a method of identifying anIL-6 binding compound, comprising (i) immobilizing IL-6 to a support,(ii) contacting said immobilized IL-6 simultaneously or in a step-wisemanner with at least one tagged or labelled binding member according tothe invention and one or more untagged or unlabelled test bindingcompounds, and (iii) identifying a new IL-6 binding compound byobserving a decrease in the amount of bound tag from the tagged bindingmember. Such methods can be performed in a high-throughput manner usinga multiwell or array format. Such assays may be also be performed insolution. See, for instance, U.S. Pat. No. 5,814,468, which is hereinincorporated by reference in its entirety. As described above, detectionof binding may be interpreted directly by the person performing themethod, for instance, by visually observing a detectable label, or adecrease in the presence thereof. Alternatively, the binding methods ofthe invention may produce a report in the form of an autoradiograph, aphotograph, a computer printout, a flow cytometry report, a graph, achart, a test tube or container or well containing the result, or anyother visual or physical representation of a result of the method.

Competition assays can also be used in epitope mapping. In one instanceepitope mapping may be used to identify the epitope bound by an IL-6binding member which optionally may have optimized neutralizing and/ormodulating characteristics. Such an epitope can be linear orconformational. A conformational epitope can comprise at least twodifferent fragments of IL-6, wherein said fragments are positioned inproximity to each other when IL-6 is folded in its tertiary orquaternary structure to form a conformational epitope which isrecognized by an inhibitor of IL-6, such as an IL-6-binding member. Intesting for competition a peptide fragment of the antigen may beemployed, especially a peptide including or consisting essentially of anepitope of interest. A peptide having the epitope sequence plus one ormore amino acids at either end may be used. Binding members according tothe present invention may be such that their binding for antigen isinhibited by a peptide with or including the sequence given.

The present invention further provides an isolated nucleic acid encodinga binding member of the present invention. Nucleic acid may include DNAand/or RNA. In one, the present invention provides a nucleic acid thatcodes for a CDR or set of CDRs or VH domain or VL domain or antibodyantigen-binding site or antibody molecule, e.g. scFv or IgG1, of theinvention as defined above.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell thatcomprises one or more constructs as above. A nucleic acid encoding anyCDR or set of CDRs or VH domain or VL domain or antibody antigen-bindingsite or antibody molecule, e.g. scFv or IgG1 as provided, itself formsan aspect of the present invention, as does a method of production ofthe encoded product, which method comprises expression from encodingnucleic acid therefor. Expression may conveniently be achieved byculturing under appropriate conditions recombinant host cells containingthe nucleic acid. Following production by expression a VH or VL domain,or binding member may be isolated and/or purified using any suitabletechnique, then used as appropriate.

Nucleic acid according to the present invention may comprise DNA or RNAand may be wholly or partially synthetic. Reference to a nucleotidesequence as set out herein encompasses a DNA molecule with the specifiedsequence, and encompasses a RNA molecule with the specified sequence inwhich U is substituted for T, unless context requires otherwise.

A yet further aspect provides a method of production of an antibody VHvariable domain, the method including causing expression from encodingnucleic acid. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

Analogous methods for production of VL variable domains and bindingmembers comprising a VH and/or VL domain are provided as further aspectsof the present invention.

A method of production may comprise a step of isolation and/orpurification of the product. A method of production may compriseformulating the product into a composition including at least oneadditional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, filamentous fungi, yeast andbaculovirus systems and transgenic plants and animals.

The expression of antibodies and antibody fragments in prokaryotic cellsis well established in the art. For a review, see for example Pluckthun[103]. A common bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of a binding member [104,105, 106]. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 ratmyeloma cells, human embryonic kidney cells, human embryonic retinacells and many others.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids e.g.phagemid, or viral e.g. ‘phage, as appropriate [107]. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Ausubel et al. [108].

A further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. Such a host cell may be invitro and may be in culture. Such a host cell may be in vivo. In vivopresence of the host cell may allow intra-cellular expression of thebinding members of the present invention as “intrabodies” orintra-cellular antibodies. Intrabodies may be used for gene therapy.

A still further aspect provides a method comprising introducing nucleicacid of the invention into a host cell. The introduction may employ anyavailable technique. For eukaryotic cells, suitable techniques mayinclude calcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using retrovirus orother virus, e.g. vaccinia or, for insect cells, baculovirus.Introducing nucleic acid in the host cell, in particular a eukaryoticcell may use a viral or a plasmid based system. The plasmid system maybe maintained episomally or may be incorporated into the host cell orinto an artificial chromosome. Incorporation may be either by random ortargeted integration of one or more copies at single or multiple loci.For bacterial cells, suitable techniques may include calcium chloridetransformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene. The purification of the expressed product may beachieved by methods known to one of skill in the art.

Nucleic acid of the invention may be integrated into the genome (e.g.chromosome) of the host cell. Integration may be promoted by inclusionof sequences that promote recombination with the genome, in accordancewith standard techniques.

The present invention also provides a method that comprises using aconstruct as stated above in an expression system in order to express abinding member or polypeptide as above.

There is evidence for involvement of IL-6 in a variety of disorders, asdiscussed elsewhere herein. The binding members of the present inventionmay therefore be used in a method of diagnosis or treatment of adisorder associated with IL-6. Such a disorder may for example be aninflammatory and/or autoimmune disorder such as for example, rheumatoidarthritis, osteoarthritis, cachexia, chronic obstructive pulmonarydisease, Juvenile idiopathic arthritis, asthma, systemic lupuserythematosus, inflammatory bowel disease, Crohn's disease oratherosclerosis. A binding member of the present invention may also beused to treat a disorder such as a tumour and/or cancer.

Binding members of the present invention may also be used in method ofdiagnosis or treatment of at least one IL-6 related disease, in apatient, animal, organ, tissue or cell, including, but not limited to:—

(the respiratory tract) obstructive airways diseases including chronicobstructive pulmonary disease (COPD); asthma, such as bronchial,allergic, intrinsic, extrinsic and dust asthma, particularly chronic orinveterate asthma (e.g. late asthma and airways hyper-responsiveness);bronchitis; acute-, allergic-, atrophic rhinitis and chronic rhinitisincluding rhinitis caseosa, hypertrophic rhinitis, rhinitis purulenta,rhinitis sicca and rhinitis medicamentosa; membranous rhinitis includingcroupous, fibrinous and pseudomembranous rhinitis and scrofoulousrhinitis; seasonal rhinitis including rhinitis nervosa (hay fever) andvasomotor rhinitis, sinusitis, idiopathic pulmonary fibrosis (IPF);sarcoidosis, farmer's lung and related diseases, adult respiratorydistress syndrome, hypersensitivity pneumonitis, fibroid lung andidiopathic interstitial pneumonia;

(bone and joints) rheumatoid arthritis, juvenile chronic arthritis,systemic onset juvenile arthritis, seronegative spondyloarthropathies(including ankylosing spondylitis, psoriatic arthritis and Reiter'sdisease), Behcet's disease, Siogren's syndrome and systemic sclerosis,gout, osteoporosis and osteoarthritis;

(skin) psoriasis, atopical dermatitis, contact dermatitis and othereczmatous dermatoses, allergic contact dermatitis, seborrhoeticdermatitis, Lichen planus, scleroderma, Pemphigus, bullous pemphigoid,Epidermolysis bullosa, urticaria, angiodermas, vasculitides, erythemas,cutaneous eosinophilias, uveitis, Alopecia areata, allergicconjunctivitis and vernalvemal conjunctivitis;

(gastrointestinal tract) gastric ulcer, Coeliac disease, proctitis,eosinopilic gastro-enteritis, mastocytosis, inflammatory bowel disease,Crohn's disease, ulcerative colitis, antiphospholipid syndrome)),food-related allergies which have effects remote from the gut, e.g.,migraine, rhinitis and eczema;

(other tissues and systemic disease) cachexia, multiple sclerosis,atherosclerosis, Acquired Immunodeficiency Syndrome (AIDS), mesangialproliferative glomerulonephritis, nephrotic syndrome, nephritis,glomerular nephritis, acute renal failure, hemodialysis, uremia,localised or discoid lupus erythematosus, systemic lupus erythematosus,Castleman's Disease, Hashimoto's thyroiditis, myasthenia gravis, type Idiabetes, type B insulin-resistant diabetes, sickle cell anaemia,iridocyclitis/uveitis/optic neuritis, nephritic syndrome, eosinophiliafascitis, hyper IgE syndrome, systemic vasculitis/wegener'sgranulomatosis, orchitis/vasectomy reversal procedures, lepromatousleprosy, alcohol-induced hepatitis, sezary syndrome and idiopathicthrombocytopenia purpura; post-operative adhesions, nephrosis, systemicinflammatory response syndrome, sepsis syndrome, gram positive sepsis,gram negative sepsis, culture negative sepsis, fungal sepsis,neutropenic fever, acute pancreatitis, urosepsis, Graves disease,Raynaud's disease, antibody-mediatated cytotoxicity, type IIIhypersensitivity reactions, POEMS syndrome (polyneuropathy,organomegaly, endocrinopathy, monoclonal gammopathy, and skin changessyndrome), mixed connective tissue disease, idiopathic Addison'sdisease, diabetes mellitus, chronic active hepatitis, primary billiarycirrhosis, vitiligo, post-MI (cardiotomy) syndrome, type IVhypersensitivity, granulomas due to intracellular organisms, Wilson'sdisease, hemachromatosis, alpha-I-antitrypsin deficiency, diabeticretinopathy, hashimoto's thyroiditis, hypothalamic-pituitary-adrenalaxis evaluation, thyroiditis, encephalomyelitis, neonatal chronic lungdisease, familial hematophagocytic lymphohistiocytosis, alopecia,radiation therapy (e.g., including but not limited to asthenia, anemia,cachexia, and the like), chronic salicylate intoxication, sleep apnea,obesity, heart failure, and meningococcemia;

(allograft rejection) acute and chronic following, for example,transplantation of kidney, heart, liver, lung, pancreas, bone marrow,bone, small bowel, skin, cartilage and cornea; and chronic graft versushost disease;

(malignant disease) leukaemia, acute lymphoblastic leukaemia (ALL),acute leukaemia, T-cell, B-cell, or FAB ALL, chromic myelocyticleukaemia (CML), acute myeloid leukaemia (AML), chronic lymphocyticleukaemia (CLL), hairy cell leukaemia, myelodyplastic syndrome (MDS),any lymphoma, Hodgkin's disease, non-hodgkin's lymphoma, any malignantlymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, renalcell carcinoma, colorectal carcinoma, prostatic carcinoma, pancreaticcarcinoma, nasopharyngeal carcinoma, malignant histiocytosis,paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors,adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastaticdisease, cancer related bone resorption, cancer related bone pain; thesuppression of cancer metastasis; the amelioration of cancer cachexia;

Cystic fibrosis, stroke, re-perfusion injury in the heart, brain,peripheral limbs and other organs;

Burn wounds, trauma/haemorrhage, ionizing radiation exposure, chronicskin ulcers;

Reproductive Diseases (e.g. Disorders of ovulation, menstruation andimplantation, pre-term labour, pre-eclampsia, endometriosis);(Infections) acute or chronic bacterial infection, acute and chronicparasitic or infectious processes, including bacterial, viral and fungalinfections, HIV infection/HIV neuropathy, meningitis, hepatitis (A, B orC, or other viral hepatitis the like), septic arthritis, peritonitis,pneumonia, epiglottitis, e. coli 0157:h7, hemolytic uremicsyndrome/thrombotic thrombocytopenic purpura, malaria, denguehemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome,streptococcal myositis, gas gangrene, Mycobacterium tuberculosis,Mycobacterium avium intracellulare, Pneumocystis carinii pneumonia,pelvic inflammatory disease, orchitis/epidydimitis, legionella, Lymedisease, influenza a, epstein-barr virus, vital-associatedhemaphagocytic syndrome, viral encephalitis/aseptic meningitis, and thelike.

Accordingly, the invention provides a method of treating an IL-6 relateddisorder, comprising administering to a patient in need thereof aneffective amount of one or more binding members of the present inventionalone or in a combined therapeutic regimen with another appropriatemedicament known in the art or described herein.

Evidence for involvement of IL-6 in certain disorders is summarisedelsewhere herein. In addition, the data presented herein furtherindicates that binding members of the invention can be used to treatsuch disorders, including preventative treatment and reduction ofseverity of the disorders. Accordingly, the invention provides a methodof treating or reducing the severity of at least one symptom of any ofthe disorders mentioned herein, comprising administering to a patient inneed thereof an effective amount of one or more binding members of thepresent invention alone or in a combined therapeutic regimen withanother appropriate medicament known in the art or described herein suchthat the severity of at least one symptom of any of the above disordersis reduced.

Thus, the binding members of the present invention are useful astherapeutic agents in the treatment of diseases or disorders involvingIL-6 and/or IL-6Ra expression and/or activity, especially aberrantexpression/activity. A method of treatment may comprise administering aneffective amount of a binding member of the invention to a patient inneed thereof, wherein aberrant expression and/or activity of IL-6 and/orIL-6Ra is decreased. A method of treatment may comprise (i) identifyinga patient demonstrating aberrant IL-6:IL-6Ra levels or activity, forinstance using the diagnostic methods described above, and (ii)administering an effective amount of a binding member of the inventionto the patient, wherein aberrant expression and/or activity of IL-6Raand/or IL-6 is decreased. An effective amount according to the inventionis an amount that decreases the aberrant expression and/or activity ofIL-6 and/or IL-6Ra so as to decrease or lessen the severity of at leastone symptom of the particular disease or disorder being treated, but notnecessarily cure the disease or disorder.

The invention also provides a method of antagonising at least one effectof IL-6, comprising contacting with or administering an effective amountof one or more binding members of the present invention such that saidat least one effect of IL-6 is antagonised. Effects of IL-6 that may beantagonised by the methods of the invention include IL-6 binding togp130, and downstream effects that arise as a consequence of thisbinding. Accordingly, further aspects of the invention provide methodsof treatment comprising administration of a binding member as provided,pharmaceutical compositions comprising such a binding member, and use ofsuch a binding member in the manufacture of a medicament foradministration, for example in a method of making a medicament orpharmaceutical composition comprising formulating the binding memberwith a pharmaceutically acceptable excipient. A pharmaceuticallyacceptable excipient may be a compound or a combination of compoundsentering into a pharmaceutical composition not provoking secondaryreactions and which allows, for example, facilitation of theadministration of the active compound(s), an increase in its lifespanand/or in its efficacy in the body, an increase in its solubility insolution or else an improvement in its conservation. Thesepharmaceutically acceptable vehicles are well known and will be adaptedby the person skilled in the art as a function of the nature and of themode of administration of the active compound(s) chosen.

Binding members of the present invention will usually be administered inthe form of a pharmaceutical composition, which may comprise at leastone component in addition to the binding member. Thus pharmaceuticalcompositions according to the present invention, and for use inaccordance with the present invention, may comprise, in addition toactive ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, inhaled, intra-tracheal, topical, intra-vesicular or byinjection, as discussed below.

Pharmaceutical compositions for oral administration, such as for examplesingle domain antibody molecules (e.g. “Nanobodies™”) etc. are alsoenvisaged in the present invention. Such oral formulations may be intablet, capsule, powder, liquid or semi-solid form. A tablet maycomprise a solid carrier, such as gelatin or an adjuvant. Liquidpharmaceutical compositions generally comprise a liquid carrier, such aswater, petroleum, animal or vegetable oils, mineral oil or syntheticoil. Physiological saline solution, dextrose or other saccharidesolution or glycols, such as ethylene glycol, propylene glycol orpolyethylene glycol may be included.

For intra-venous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles, suchas Sodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be employed as required including buffers such asphosphate, citrate and other organic acids; antioxidants, such asascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecularweight polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagines, histidine,arginine, or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose or dextrins; chelating agents,such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions, such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG).

Binding members of the present invention may be formulated in liquid,semi-solid or solid forms depending on the physicochemical properties ofthe molecule and the route of delivery. Formulations may includeexcipients, or combinations of excipients, for example: sugars, aminoacids and surfactants. Liquid formulations may include a wide range ofantibody concentrations and pH. Solid formulations may be produced bylyophilisation, spray drying, or drying by supercritical fluidtechnology, for example. Formulations of binding members will dependupon the intended route of delivery: for example, formulations forpulmonary delivery may consist of particles with physical propertiesthat ensure penetration into the deep lung upon inhalation; topicalformulations (e.g. for treatment of scarring, e.g. dermal scarring) mayinclude viscosity modifying agents, which prolong the time that the drugis resident at the site of action. A binding member may be prepared witha carrier that will protect the binding member against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are known to those skilled in the art [109].

Treatment may be given orally (such as for example single domainantibody molecules (e.g. “Nanobodies™”)) by injection (for example,subcutaneously, intra-articular, intra-venously, intra-peritoneal,intra-arterial or intra-muscularly), by inhalation, intra-tracheal, bythe intra-vesicular route (instillation into the urinary bladder), ortopically (for example intra-ocular, intra-nasal, rectal, into wounds,on skin). The treatment may be administered by pulse infusion,particularly with declining doses of the binding member. The route ofadministration can be determined by the physicochemical characteristicsof the treatment, by special considerations for the disease or by therequirement to optimize efficacy or to minimize side-effects. Oneparticular route of administration is intra-venous. Another route ofadministering pharmaceutical compositions of the present invention issubcutaneously. It is envisaged that treatment will not be restricted touse in the clinic. Therefore, subcutaneous injection using a needle-freedevice is also advantageous.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

A binding member of the invention may be used as part of a combinationtherapy in conjunction with an additional medicinal component.Combination treatments may be used to provide significant synergisticeffects, particularly the combination of a binding member of theinvention with one or more other drugs. A binding member of theinvention may be administered concurrently or sequentially or as acombined preparation with another therapeutic agent or agents, for thetreatment of one or more of the conditions listed herein.

A binding member of the invention may be used as a chemosensitiserwhereby it can increase therapeutic efficacy of cytotoxic agents, andmay thus be provided for administration in combination with one or morecytotoxic agents, either simultaneously or sequentially. The bindingmember may also be used as a radio sensitiser whereby it can improveefficacy of radiation, and may thus be provided for administration incombination with radiation, either simultaneously or sequentially.

A binding member according to the present invention may be provided incombination or addition with one or more of the following agents:

-   -   a cytokine or agonist or antagonist of cytokine function (e.g.        an agent which acts on cytokine signalling pathways, such as a        modulator of the SOCS system), such as an alpha-, beta- and/or        gamma-interferon; insulin-like growth factor type I (IGF-1), its        receptors and associated binding proteins; interleukins (IL),        e.g. one or more of IL-1 to -33, and/or an interleukin        antagonist or inhibitor, such as anakinra; inhibitors of        receptors of interleukin family members or inhibitors of        specific subunits of such receptors, a tumour necrosis factor        alpha (TNF-α) inhibitor, such as an anti-TNF monoclonal        antibodies (for example infliximab, adalimumab and/or CDP-870)        and/or a TNF receptor antagonist, e.g. an immunoglobulin        molecule (such as etanercept) and/or a low-molecular-weight        agent, such as pentoxyfylline;    -   a modulator of B cells, e.g. a monoclonal antibody targeting        B-lymphocytes (such as CD20 (rituximab) or MRA-aIL16R) or        1-lymphocytes (e.g. CTLA4-Ig, HuMax 11-15 or Abatacept);    -   a modulator that inhibits osteoclast activity, for example an        antibody to RANKL; a modulator of chemokine or chemokine        receptor function, such as an antagonist of CCR1, CCR2, CCR2A,        CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11        (for the C—C family); CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 and        CXCR6 (for the C—X—C family) and CX3CR1 for the C—X₃—C family;    -   an inhibitor of matrix metalloproteases (MMPs), i.e. one or more        of the stromelysins, the collagenases and the gelatinases as        well as aggrecanase, especially collagenase-1 (MMP-1),        collagenase-2 (MMP-8), collagenase-3 (MMP-13), stromelysin-1        (MMP-3), stromelysin-2 (MMP-10) and/or stromelysin-3 (MMP-11)        and/or MMP-9 and/or MMP-12, e.g. an agent such as doxycycline;    -   a leukotriene biosynthesis inhibitor, 5-lipoxygenase (5-LO)        inhibitor or 5-lipoxygenase activating protein (FLAP)        antagonist, such as zileuton; ABT-761; fenleuton; tepoxalin;        Abbott-79175; Abbott-85761;        N-(5-substituted)-thiophene-2-alkylsulfonamides;        2,6-di-tert-butylphenolhydrazones; methoxytetrahydropyrans such        as Zeneca ZD-2138; the compound SB-210661; a        pyridinyl-substituted 2-cyanonaphthalene compound, such as        L-739,010; a 2-cyanoquinoline compound, such as L-746,530;        indole and/or a quinoline compound, such as MK-591, MK-886        and/or BAY×1005;    -   a receptor antagonist for leukotrienes (LT) B4, LTC4, LTD4, and        LTE4, selected from the group consisting of the        phenothiazin-3-1s, such as L-651,392; amidino compounds, such as        CGS-25019c; benzoxalamines, such as ontazolast;        benzenecarboximidamides, such as BITL 284/260; and compounds,        such as zafirlukast, ablukast, montelukast, pranlukast,        verlukast (MK-679), RG-12525, Ro-245913, iralukast (CGP 45715A)        and BAY×7195;    -   a phosphodiesterase (PDE) inhibitor, such as a methylxanthanine,        e.g. theophylline and/or aminophylline; and/or a selective PDE        isoenzyme inhibitor, e.g. a PDE4 inhibitor and/or inhibitor of        the isoform PDE4D and/or an inhibitor of PDE5; a histamine type        1 receptor antagonist, such as cetirizine, loratadine,        desloratadine, fexofenadine, acrivastine, terfenadine,        astemizole, azelastine, levocabastine, chlorpheniramine,        promethazine, cyclizine, and/or mizolastine (generally applied        orally, topically or parenterally);    -   a proton pump inhibitor (such as omeprazole) or gastroprotective        histamine type 2 receptor antagonist;    -   an antagonist of the histamine type 4 receptor;    -   an alpha-1/alpha-2 adrenoceptor agonist vasoconstrictor        sympathomimetic agent, such as propylhexedrine, phenylephrine,        phenylpropanolamine, ephedrine, pseudoephedrine, naphazoline        hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline        hydrochloride, xylometazoline hydrochloride, tramazoline        hydrochloride and ethylnorepinephrine hydrochloride;    -   an anticholinergic agent, e.g. a muscarinic receptor (M1, M2,        and M3) antagonist, such as atropine, hyoscine, glycopyrrrolate,        ipratropium bromide, tiotropium bromide, oxitropium bromide,        pirenzepine and telenzepine;    -   a beta-adrenoceptor agonist (including beta receptor subtypes        1-4), such as isoprenaline, salbutamol, formoterol, salmeterol,        terbutaline, orciprenaline, bitolterol mesylate and/or        pirbuterol, e.g. a chiral enantiomer thereof;    -   a chromone, e.g. sodium cromoglycate and/or nedocromil sodium;    -   a glucocorticoid, such as flunisolide, triamcinolone acetonide,        beclomethasone dipropionate, budesonide, fluticasone propionate,        ciclesonide, and/or mometasone furoate;    -   an agent that modulate nuclear hormone receptors, such as a        PPAR;    -   an immunoglobulin (Ig) or Ig preparation or an antagonist or        antibody modulating Ig function, such as anti-IgE (e.g.        omalizumab);    -   other systemic or topically-applied anti-inflammatory agent,        e.g. thalidomide or a derivative thereof, a retinoid, dithranol        and/or calcipotriol;    -   combinations of aminosalicylates and sulfapyridine, such as        sulfasalazine, mesalazine, balsalazide, and olsalazine; and        immunomodulatory agents, such as the thiopurines; and        corticosteroids, such as budesonide;    -   an antibacterial agent, e.g. a penicillin derivative, a        tetracycline, a macrolide, a beta-lactam, a fluoroquinolone,        metronidazole and/or an inhaled aminoglycoside; and/or an        antiviral agent, e.g. acyclovir, famciclovir, valaciclovir,        ganciclovir, cidofovir; amantadine, rimantadine; ribavirin;        zanamavir and/or oseltamavir; a protease inhibitor, such as        indinavir, nelfinavir, ritonavir and/or saquinavir; a nucleoside        reverse transcriptase inhibitor, such as didanosine, lamivudine,        stavudine, zalcitabine, zidovudine; a non-nucleoside reverse        transcriptase inhibitor, such as nevirapine, efavirenz;    -   a cardiovascular agent, such as a calcium channel blocker,        beta-adrenoceptor blocker, angiotensin-converting enzyme (ACE)        inhibitor, angiotensin-2 receptor antagonist; lipid lowering        agent, such as a statin and/or fibrate; a modulator of blood        cell morphology, such as pentoxyfylline; a thrombolytic and/or        an anticoagulant, e.g. a platelet aggregation inhibitor;    -   a CNS agent, such as an antidepressant (such as sertraline),        anti-Parkinsonian drug (such as deprenyl, L-dopa, ropinirole,        pramipexole; MACS inhibitor, such as selegine and rasagiline;        comP inhibitor, such as tasmar; A-2 inhibitor, dopamine reuptake        inhibitor, NMDA antagonist, nicotine agonist, dopamine agonist        and/or inhibitor of neuronal nitric oxide synthase) and an        anti-Alzheimer's drug, such as donepezil, rivastigmine, tacrine,        COX-2 inhibitor, propentofylline or metrifonate;    -   an agent for the treatment of acute and chronic pain, e.g. a        centrally or peripherally-acting analgesic, such as an opioid        analogue or derivative, carbamazepine, phenytoin, sodium        valproate, amitryptiline or other antidepressant agent,        paracetamol, or non-steroidal anti-inflammatory agent;    -   a parenterally or topically-applied (including inhaled) local        anaesthetic agent, such as lignocaine or an analogue thereof;    -   an anti-osteoporosis agent, e.g. a hormonal agent, such as        raloxifene, or a biphosphonate, such as alendronate;    -   (i) a tryptase inhibitor; (ii) a platelet activating factor        (PAF) antagonist; (iii) an interleukin converting enzyme (ICE)        inhibitor; (iv) an IMPDH inhibitor; (v) an adhesion molecule        inhibitors including VLA-4 antagonist; (vi) a cathepsin; (vii) a        kinase inhibitor, e.g. an inhibitor of tyrosine kinases (such as        Btk, Itk, Jak3 MAP examples of inhibitors might include        Gefitinib, Imatinib mesylate), a serine/threonine kinase (e.g.        an inhibitor of MAP kinase, such as p38, JNK, protein kinases A,        B and C and IKK), or a kinase involved in cell cycle regulation        (e.g. a cylin dependent kinase); (viii) a glucose-6 phosphate        dehydrogenase inhibitor; (ix) a kinin-B.subl.—and/or B.sub2.        -receptor antagonist; (x) an anti-gout agent, e.g.        colchicine; (xi) a xanthine oxidase inhibitor, e.g.        allopurinol; (xii) a uricosuric agent, e.g. probenecid,        sulfinpyrazone, and/or benzbromarone; (xiii) a growth hormone        secretagogue; (xiv) transforming growth factor (TGF(3); (xv)        platelet-derived growth factor (PDGF); (xvi) fibroblast growth        factor, e.g. basic fibroblast growth factor (bFGF); (xvii)        granulocyte macrophage colony stimulating factor        (GM-CSF); (xviii) capsaicin cream; (xix) a tachykinin NK.subl.        and/or NK.sub3. receptor antagonist, such as NKP-608C, SB-233412        (talnetant) and/or D-4418; (xx) an elastase inhibitor, e.g.        UT-77 and/or ZD-0892; (xxi) a TNF-alpha converting enzyme        inhibitor (TACE); (xxii) induced nitric oxide synthase (iNOS)        inhibitor or (xxiii) a chemoattractant receptor-homologous        molecule expressed on TH2 cells (such as a CRTH2        antagonist); (xxiv) an inhibitor of a P38 (xxv) agent modulating        the function of Toll-like receptors (TLR) and (xxvi) an agent        modulating the activity of purinergic receptors, such as        P2X7; (xxvii) an inhibitor of transcription factor activation,        such as NFkB, API, and/or STATS.

An inhibitor may be specific or may be a mixed inhibitor, e.g. aninhibitor targeting more than one of the molecules (e.g. receptors) ormolecular classes mentioned above.

The binding member could also be used in association with achemotherapeutic agent or another tyrosine kinase inhibitor inco-administration or in the form of an immunoconjugate. Fragments ofsaid antibody could also be use in bispecific antibodies obtained byrecombinant mechanisms or biochemical coupling and then associating thespecificity of the above described antibody with the specificity ofother antibodies able to recognize other molecules involved in theactivity for which IL-6 is associated.

For treatment of an inflammatory disease, a binding member of theinvention may be combined with one or more agents, such as non-steroidalanti-inflammatory agents (hereinafter NSAIDs) including non-selectivecyclo-oxygenase (COX)-1/COX-2 inhibitors whether applied topically orsystemically, such as piroxicam, diclofenac, propionic acids, such asnaproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates,such as mefenamic acid, indomethacin, sulindac, azapropazone,pyrazolones, such as phenylbutazone, salicylates, such as aspirin);selective COX-2 inhibitors (such as meloxicam, celecoxib, rofecoxib,valdecoxib, lumarocoxib, parecoxib and etoricoxib); cyclo-oxygenaseinhibiting nitric oxide donors (CINODs); glucocorticosteroids (whetheradministered by topical, oral, intra-muscular, intra-venous orintra-articular routes); methotrexate, leflunomide; hydroxychloroquine,d-penicillamine, auranofin or other parenteral or oral goldpreparations; analgesics; diacerein; intra-articular therapies, such ashyaluronic acid derivatives; and nutritional supplements, such asglucosamine.

A binding member of the invention can also be used in combination withan existing therapeutic agent for the treatment of cancer. Suitableagents to be used in combination include:

-   -   (i) antiproliferative/antineoplastic drugs and combinations        thereof, as used in medical oncology, such as Gleevec (imatinib        mesylate), alkylating agents (for example cis-platin,        carboplatin, cyclophosphamide, nitrogen mustard, melphalan,        chlorambucil, busulphan and nitrosoureas); antimetabolites (for        example antifolates, such as fluoropyrimidines like        5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine        arabinoside, hydroxyurea, gemcitabine and paclitaxel);        antitumour antibiotics (for example anthracyclines like        adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin,        idarubicin, mitomycin-C, dactinomycin and mithramycin);        antimitotic agents (for example vinca alkaloids like        vincristine, vinblastine, vindesine and vinorelbine and taxoids        like taxol and taxotere); and topoisomerase inhibitors (for        example epipodophyllotoxins like etoposide and teniposide,        amsacrine, topotecan and camptothecins); (ii) cytostatic agents,        such as antioestrogens (for example tamoxifen, toremifene,        raloxifene, droloxifene and iodoxyfene), oestrogen receptor down        regulators (for example fulvestrant), antiandrogens (for example        bicalutamide, flutamide, nilutamide and cyproterone acetate),        LHRH antagonists or LHRH agonists (for example goserelin,        leuprorelin and buserelin), progestogens (for example megestrol        acetate), aromatase inhibitors (for example as anastrozole,        letrozole, vorazole and exemestane) and inhibitors of        5α-reductase, such as finasteride;    -   (iii) Agents which inhibit cancer cell invasion (for example        metalloproteinase inhibitors like marimastat and inhibitors of        urokinase plasminogen activator receptor function);    -   (iv) inhibitors of growth factor function, for example such        inhibitors include growth factor antibodies, growth factor        receptor antibodies (for example the anti-erbb2 antibody        trastuzumab and the anti-erbb1 antibody cetuximab [C225]),        farnesyl transferase inhibitors, tyrosine kinase inhibitors and        serine/threonine kinase inhibitors, for example inhibitors of        the epidermal growth factor family (for example EGFR family        tyrosine kinase inhibitors, such as        N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)        quinazolin-4-amine (gefitinib, AZD1839),        N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine        (erlotinib, OSI-774) and        6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine        (CI 1033)), for example inhibitors of the platelet-derived        growth factor family and for example inhibitors of the        hepatocyte growth factor family;    -   (v) antiangiogenic agents, such as those which inhibit the        effects of vascular endothelial growth factor (for example the        anti-vascular endothelial cell growth factor antibody        bevacizumab, compounds, such as those disclosed in International        Patent Applications WO 97/22596, WO 97/30035, WO 97/32856 and WO        98/13354, each of which is incorporated herein in its entirety)        and compounds that work by other mechanisms (for example        linomide, inhibitors of integrin αvβ3 function and angiostatin);    -   (vi) vascular damaging agents, such as combretastatin A4 and        compounds disclosed in International Patent Applications WO        99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and        WO 02/08213 (each of which is incorporated herein in its        entirety);    -   (vii) antisense therapies, for example those which are directed        to the targets listed above, such as ISIS 2503, an anti-ras        antisense;    -   (viii) gene therapy approaches, including for example approaches        to replace aberrant genes, such as aberrant p53 or aberrant        BRCA1 or BRCA2, GDEPT (gene directed enzyme pro-drug therapy)        approaches, such as those using cytosine deaminase, thymidine        kinase or a bacterial nitroreductase enzyme and approaches to        increase patient tolerance to chemotherapy or radiotherapy, such        as multi-drug resistance gene therapy; and    -   (ix) immunotherapeutic approaches, including for example ex vivo        and in vivo approaches to increase the immunogenicity of patient        tumour cells, such as transfection with cytokines, such as        interleukin 2, interleukin 4 or granulocyte macrophage colony        stimulating factor, approaches to decrease T-cell anergy,        approaches using transfected immune cells, such as        cytokine-ransfected dendritic cells, approaches using        cytokine-transfected tumour cell lines and approaches using        anti-idiotypic antibodies.

A binding member of the invention and one or more of the aboveadditional medicinal components may be used in the manufacture of amedicament. The medicament may be for separate or combinedadministration to an individual, and accordingly may comprise thebinding member and the additional component as a combined preparation oras separate preparations. Separate preparations may be used tofacilitate separate and sequential or simultaneous administration, andallow administration of the components by different routes e.g. oral andparenteral administration.

In accordance with the present invention, compositions provided may beadministered to mammals. Administration is normally in a“therapeutically effective amount”, this being sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated, the particular mammal being treated, the clinicalcondition of the individual patient, the cause of the disorder, the siteof delivery of the composition, the type of binding member, the methodof administration, the scheduling of administration and other factorsknown to medical practitioners. Prescription of treatment, e.g.decisions on dosage etc, is within the responsibility of generalpractitioners and other medical doctors and may depend on the severityof the symptoms and/or progression of a disease being treated.Appropriate doses of antibody are well known in the art [110, 111].Specific dosages indicated herein or in the Physician's Desk Reference(2003) as appropriate for the type of medicament being administered maybe used. A therapeutically effective amount or suitable dose of abinding member of the invention can be determined by comparing its invitro activity and in vivo activity in an animal model. Methods forextrapolation of effective dosages in mice and other test animals tohumans are known. The precise dose will depend upon a number of factors,including whether the antibody is for diagnosis, prevention or fortreatment, the size and location of the area to be treated, the precisenature of the antibody (e.g. whole antibody, fragment or diabody) andthe nature of any detectable label or other molecule attached to theantibody. A typical antibody dose will be in the range 100 μg to 1 g forsystemic applications, and 1 μg to 1 mg for topical applications. Aninitial higher loading dose, followed by one or more lower doses, may beadministered. Typically, the antibody will be a whole antibody, e.g. theIgG1 isotype. This is a dose for a single treatment of an adult patient,which may be proportionally adjusted for children and infants, and alsoadjusted for other antibody formats in proportion to molecular weight.Treatments may be repeated at daily, twice-weekly, weekly or monthlyintervals, at the discretion of the physician. Treatments may be everytwo to four weeks for subcutaneous administration and every four toeight weeks for intra-venous administration. Treatment may be periodic,and the period between administrations is about two weeks or more, e.g.about three weeks or more, about four weeks or more, or about once amonth. Treatment may be given before, and/or after surgery, and/or maybe administered or applied directly at the anatomical site of surgicaltreatment.

IL-6 binding members of the invention may offer advantages in terms ofdosage and administration requirements, compared with antibodies tosIL-6Ra. As noted elsewhere herein, circulating levels of IL-6 aresignificantly lower than circulating levels of sIL-6Ra in disease.Accordingly, use of an IL-6 binding member, as opposed to an anti-IL-6Rbinding member, has significant advantages in that the amount of drug tobe manufactured for each dose to patients may be lower. Also if the doseof an anti-IL6 therapeutic is lower there may be significant advantagesin that the low dose facilitates sub-cutaneous injections as well asintra-venous (i.v.) injections. It is well known to those skilled in theart that sub-cutaneous dosing may be limited by the amount of bindingmember, e.g. antibody molecule, required per dose. This is due to thesub-cutaneous injections being limited by the volume that can beinjected at one site in the skin. Subcutaneous injection volumes of 1.2ml or less are typically utilised. As it may be increasingly difficultto formulate a binding member for sub-cutaneous injection atconcentrations greater than 50 mg/ml, doses above 100 mg via this routeusually require multiple injections and more discomfort for the patient.

Having a lower dose anti-IL-6 therapeutic may also require a lower“loading” dose of antibody to inhibit all the systemic IL-6 comparedwith the systemic sIL-6Ra as this is at higher concentrations.

Further benefits may be associated with targeting IL-6 rather than 5IL-6 receptor, representing additional advantages of binding members ofthe invention as compared with binding members for IL-6Ra.

For example, there are literature reports which show that thecirculating levels of IL-6 are significantly lower than circulatinglevels of sIL-6Ra in disease [112, 113]. As the levels of sIL-6R aresignificantly higher than IL-6 levels, more anti-sIL-6R binding membermay be required to neutralise the sIL-6Ra, compared with the amount ofanti-IL-6 binding member required to neutralise IL-6. Hence, a lowerdose of an anti-ligand binding member may be needed, compared with if ananti-receptor binding member were used.

Targeting IL-6 ligand rather than IL-6 receptor may reduce levels ofIL-6 in disease but still allow IL-6 levels to increase duringinfection, where IL-6 is up-regulated as part of the immune response.

Kawano et al. [4] showed that IL-6 was a potent growth factor and showedthat myeloma cells freshly isolated from patients produced IL-6 andexpress its receptors. Moreover, anti-IL-6 antibody inhibits the invitro growth of myeloma cells. This is direct evidence that an autocrineloop is operating in oncogenesis of human myelomas. Subsequent to thatstudy, Van Zaanen et al. [5] demonstrated that the production of IL-6 inmultiple myeloma patients decreases when treated with an anti-IL-6ligand antibody.

A number of further studies show that IL-6 is involved in an autocrinefeedback loop in other cell types e.g. smooth muscle cells (SMC) [114],U373-MG astroglioma cells [115], 3T3 adipocytes [116], neurons [117],endothelial cells [118] and Kaposi's sarcoma cells [119]. Inhibition ofIL-6 using an anti-IL6 binding member in disease can therefore lead to adecrease in the basal disease production of IL-6.

Further, anti-IL-6 binding members bind IL-6 in the systemiccirculation, in contrast with binding members to IL-6 receptor whichneed to penetrate the tissue in order to occupy the receptor on thesurface of cells involved in the pathology of the disease to be treated.

Binding members to IL-6 may form an equilibrium with IL-6 in thesystemic circulation, having the effect of causing gradients acrossbarriers e.g. the synovial membrane, which has the net effect ofremoving active IL-6 from the joint and forming an inactive complex withthe binding member. The consequence of this is that an IL-6 bindingmember may have quicker onset and dosing regime may be different andpotentially easier to optimise, compared with an IL-6R binding member.

IL-6 signalling is mediated by IL-6 binding to IL-6R and that complexbinding to gp130. Given that IL-6 and IL-6Ra binding is of nanomolaraffinity (about 5 nM) and that IL6:1L6R complex and gp130 binding is ofpicomolar affinity, a binding member which targets IL-6 faces a loweramount of competition for IL-6 binding and so may suppress a greaterproportion of IL-6 signalling. Although this may also apply for abinding member targeting the soluble IL-6Ra and preventing IL-6:IL-6Racomplex formation, if the IL-6Ra is membrane bound then because ofsteric constraints it may be more difficult for an anti-IL-6Ra to bindand inhibit the IL-6Ra presented on the membrane.

EXAMPLES Example 1. Lead Isolation 1.1 Selections

Naïve human single chain Fv (scFv) phage display libraries cloned 5 into a phagemid vector based on the filamentous phage M13 were used forselections [120, 121]). Anti-IL-6 specific scFv antibodies were isolatedfrom the phage display libraries using a series of selection cycles onrecombinant human IL-6 essentially as previously described by Vaughan etal [120] and Hawkins et al [122]. In brief, for bio-panning selections,human IL-6 in PBS (Dulbecco's PBS, pH7.4) was adsorbed onto wells of amicrotitre plate overnight at 4° C. Wells were washed with PBS thenblocked for 1 h with PBS-Marvel (3% w/v). Purified phage in PBS-Marvel(3% w/v) were added to the wells and allowed to bind coated antigen for1 h. Unbound phage was removed by a series of wash cycles usingPBS-Tween (0.1% v/v) and PBS. Bound phage particles were eluted,infected into bacteria and rescued for the next round of selection[120].

1.2 Inhibition of IL-6 Binding to IL-6 Receptor by Crude scFv

A representative number of individual clones from the second round ofselections were grown up in 96-well plates. ScFvs were expressed in thebacterial periplasm and screened for their inhibitory activity in aHTRF® (Homogeneous Time-Resolved Fluorescence, CIS Bio international)human IL-6/human IL-6 receptor-binding assay. In this assay, samplescompeted for binding to cryptate labelled human IL-6 (R&D Systems), withbiotinylated IL-6R (Peprotech). A reference anti-IL-6 mAb (BiosourceAHC0562) was included in all potency assays as a positive control. Thedetailed assay method is provided in the Materials and Methods section.

1.3 Reformatting of scFv to IgG1

Clones were converted from scFv to IgG format by sub-cloning the VH andVL domains into vectors expressing whole antibody heavy and light chainsrespectively. The VH domain was cloned into a vector (pEU15.1)containing the human heavy chain constant domains and regulatoryelements to express whole IgG heavy chain in mammalian cells. Similarly,the VL domain was cloned into either vector pEU3.4 for the expression ofthe human kappa light chain or pEU4.4 for the expression of the humanlambda light chain constant domains, with regulatory elements to expresswhole IgG light chain in mammalian cells. Vectors for the expression ofheavy chains and light chains were originally described in ref [123].Cambridge Antibody Technology vectors have been engineered simply byintroducing an OriP element. To obtain IgGs, the heavy and light chainIgG expressing vectors were transfected into EBNA-HEK293 mammaliancells. IgGs were expressed and secreted into the medium. Harvests werepooled and filtered prior to purification. The IgG was purified usingProtein A chromatography. Culture supernatants are loaded on a column ofappropriate size of Ceramic Protein A (BioSepra) and washed with 50 mMTris-HCl pH 8.0, 250 mM NaCl. Bound IgG was eluted from the column using0.1 M Sodium Citrate (pH 3.0) and neutralised by the addition of Tris-HC1 (pH 9.0). The eluted material was buffer exchanged into PBS usingNaplO columns (Amersham, #17-0854-02) and the concentration of IgG wasdetermined spectrophotometrically using an extinction coefficient basedon the amino acid sequence of the IgG [124]. The purified IgG wereanalysed for aggregation or degradation using SEC-HPLC and by SDS-PAGE.

1.4 Inhibition of IL-6 Binding to IL-6 Receptor by Purified scFv 30 andIgG

ScFv which showed a significant inhibitory effect on the IL-6:IL-6Rinteraction as crude periplasmic extracts, were subjected to DNAsequencing [120, 125]. Unique scFvs were expressed again in bacteria andpurified by affinity chromatography (as described by Bannister et al[126]. Purified IgG samples of these clones were also prepared asdescribed in section 1.3. The potencies of these samples were determinedby competing a dilution series of the purified preparation againstbiotinylated sIL-6R for binding to HIS FLAG tagged human IL-6 (in houseE. coli derived).

The results for clone CAN022D10, as an scFv and as an IgG having a humanheavy chain and kappa light chain constant domain, are given in Table 1.Detailed protocols are provided in Materials and Methods section.

TABLE 1 Potency of CAN022D10 scFv and IgG in the receptor-ligand HTRFbiochemical assay CLONE IC50 scFv (nM) IC50 IgG (AM) CAN022D10 45 0.311.5 Inhibition of IL-6 Induced Proliferation of TF-1 Cells by PurifiedscFv, and IgG

The neutralisation potency of purified scFv preparations against humanand cynomolgus IL-6 bioactivity was assessed using TF-1 cellproliferation assay. TF-1 is a human premyeloid cell line establishedfrom a patient with erythroleukaemia [134]. The TF-1 cell line is factordependent for survival and proliferation. TF-1 cells were shown torespond to both human and cynomolgus IL-6 (in-house, E. coli derived)and were maintained in media containing human GM-CSF (4 ng/ml, R&DSystems). Inhibition of IL-6 dependent proliferation was determined bymeasuring the reduction in incorporation of tritiated thymidine into thenewly synthesized DNA of dividing cells. A detailed description of theprotocol is provided in the Materials and Methods section.

Purified scFv preparations of CAN022D10 were capable of inhibiting theIL-6 induced proliferation of the TF-1 cells at the maximumconcentration tested, although complete inhibition was not observed. Itwas therefore not possible to calculate accurate IC₅₀ potency data fromthe results obtained. When tested as a purified IgG, the IC₅₀ forCAN022D10 was calculated as being 93 nM.

1.6 Selectivity and Species Cross Reactivity of Antibodies in 5 DELFIA0Epitope Competition Assays

The species cross reactivity and selectivity of antibodies to IL-6family members was established using DELFIA® epitope competition assays,by measuring inhibition of biotinylated HIS FLAG IL-6 (in-house, E. coliderived), binding each immobilised anti-IL-6 antibody.

Titrations of purified, leukaemia inhibitory factor (LIF) (Chemicon),ciliary neurotrophic factor (CNTF), IL-11 and oncostatin M (all R & DSystems) were tested in each assay to establish the potency for eachstructurally related protein, as measured by IC₅₀ values in the assay.

Titrations of IL-6 species including cynomolgus (in house E. coliderived), human HIS FLAG IL-6 (in house HEK-EBNA derived), rat andmurine IL-6 (both R & D Systems) were tested in each assay to establishthe species cross-reactivity of the antibodies. Example results of thisexperiment are provided in Table 2. Details of the protocol are providedin the Materials and Methods section.

TABLE 2 Potencies of IL-6 related proteins and different IL-6 species inthe CAN22D10 competition assay Protein IC₅₀ (nM) Human IL-6  32*Cynomolgus IL-6 100* Murine IL-6 No inhibition Rat IL-6 No inhibitionHuman IL-11 No inhibition Human CNTF No inhibition Human LIF Noinhibition Human Oncostatin M No inhibition *Values are approximationsas incomplete curves were obtained for the samples1.7 Inhibition of Endogenous IL-6 Induced VEGF Release from HumanSynovial Fibroblast by Purified IgG

Potencies of the antibodies were evaluated for inhibition of IL-6induced VEGF release from human synovial fibroblasts explanted fromdonors with rheumatoid arthritis. A detailed protocol for this procedureis provided in Materials and Methods. In brief, titrations of the testIgG were added cultured fibroblasts, which were then stimulated by theaddition of human IL-13 and soluble human IL-6Ra to induce IL-6expression and enable signalling of the cells to induce VEGF expression.Following a 48 h incubation, supernatants were removed and tested byELISA for the expression of VEGF using a commercially available kit (R &D Systems). These data were used to determine IC₅₀ for the CAN022D10,which was calculated as being 45 nM.

Example 2 Antibody Optimisation

2.1 Identification of Amino Acids that May Improve Binding of the LeadAntibody to IL-6

A strategy to identify key residues in the parent antibody sequence thatmay improve binding to IL-6 was carried out by introducing randommutations throughout the CAN022D10 scFv sequence. This was achieved bytwo rounds of mutagenesis using A Diversify™ PCR random mutagenesis kit(BD biosciences), following the manufacturers instructions toincorporate on average, 8.1 mutations per kilobase in the nucleic acidsequence per round of mutagenesis. The selections were performedessentially as described previously (Hanes et al 2000; Methods inEnzymology, 328, 404-430). In brief, the random mutagenesis library ofthe parent clone was transcribed in to mRNA and using a process ofstalled translation, mRNA-ribosome-scFv complexes were formed. Thesecomplexes were incubated with bio-huIL-6, and those that bound to theantigen were then captured on streptavidin-coated paramagnetic beads.Non-specific ribosome complexes were washed away, and mRNA was isolatedfrom the bound ribosomal complexes, reverse transcribed to cDNA and thenamplified by PCR. This DNA was used for the next round of selectionand/or cloned out for screening. The selection process was repeated inthe presence of decreasing concentrations of bio-huIL-6 (100 nM to 0.1nM over 4 rounds). ScFv isolated by ribosome display were cloned intothe phagemid vector pCANTAB6 by Ncol/Notl restriction endonucleasedigestion (New England Biolabs) of the ribosome display construct,followed by ligation in to Ncol/Notl digested pCANTAB6 using T4 DNAligase (New England Biolabs) [127]. Ligated DNA was then transformed into chemically competent TG-1 cells, and crude scFv from individualclones were competed against CAN022D10 IgG for binding to HIS/FLAG IL-6tested in a ligand-antibody biochemical assay.

2.2 Identification of Improved Clones Using an Antibody-LigandBiochemical Assay (Using CAN022D10 IgG)

Crude scFv preparations from a representative number of individualclones for the round 3 and round 4 outputs were screened for theirinhibitory activity in a CAN022D10 IgG-IL-6 HTRF® binding assay. In thisassay, binding of biotinyated antibody and FLAG-tagged IL-6 was detectedusing cryptate labelled anti-FLAG monoclonal antibody and streptavidinXI,^(ent!)™. The detailed assay method is provided in the Materials andMethods section.

ScFv that demonstrated a significant inhibitory effect were 5 sequencedand produced as purified preparations as described in section 1.4. TheIC₅₀ value for each scFv was then calculated from data obtained by atesting dilution series of the purified sample in the HTRFantibody-ligand biochemical assay and TF-1 proliferation assay. The mostpotent clones in the TF-1 proliferation assay were converted to IgG witha heavy chain constant domain and kappa light chain constant domain, asdescribed previously, and were re-tested in the TF-1 proliferationassay. Example potency data for both purified scFv and IgG for eachsample is provided in Table 3.

TABLE 3 Examples of clones with improved potencies in theligand-antibody biochemical and TF-1 proliferation assays, isolated fromthe ribosome display CAN022D10 random mutagenesis library IC_(50 (pM))Biochemical Assay TF-1 Proliferation Assay Clone scFv IgG* scFv IgGAntibody 2 35 36 9600 16 Antibody 3 22 43 7300 50 Antibody 4 24 43 1340061 Antibody 5 65 26 12400 42 *Protocol was modified for IgG potencydetermination so scFv and IgG potencies for each clone should not bedirectly compared. For details of modifications, see Materials andMethods.

2.3 Optimisation of Parent Clone by Targeted Mutagenesis

Lead antibodies were optimised using a targeted mutagenesis approachusing affinity-based phage display selections. For the targetedmutagenesis approach, large scFv-phage libraries derived from the leadclones were created by oligonucleotide-directed mutagenesis of thevariable heavy (VH) and light (VL) chain complementarity determiningregions 3 (CDR3) using standard molecular biology techniques [128]. Thelibraries were subjected to affinity-based phage display selections inorder to select variants with higher affinity for IL-6. In consequence,these should show an improved inhibitory activity for IL-6 binding itsreceptor. The selections were performed essentially as describedpreviously [129]. In brief, the scFv phage particles were incubated withrecombinant biotinylated human IL-6 in solution (bio-huIL-6, in house E.coli derived and modified in house). ScFv-phage bound to antigen werethen captured on streptavidin-coated paramagnetic beads (Dynabeads® M280) following the manufacturer's recommendations. The selectedscFv-phageparticles were then rescued as described previously [125], andthe selection process was repeated in the presence of decreasingconcentrations of bio-huIL-6 (50 nM to 0.1 nM over 3 rounds).

Upon completion of 3 rounds of selection, the VH and VL randomisedlibraries were recombined to form a single library in which clonescontained randomly paired individually randomised VH and VL sequences.Selections were then continued as previously described in the presenceof decreasing concentrations of bio-huIL-6 (0.1 nM to 0.1 pM over afurther 4 rounds).

2.4 Identification of Improved Clones from the Targeted MutagenesisUsing an Antibody-Ligand Biochemical Assay (Using Antibody 5 IgG)

Crude scFv from clones isolated from the targeted mutagenesis selectionoutputs were tested in an antibody-ligand biochemical assay, essentiallyas described in section 2.2. For these outputs, the biochemical assaywas re-configured to use Antibody 5 IgG. This antibody is an improvedvariant on CAN02210 with greater potency in the TF-1 proliferationassay. Incorporation of this more potent IgG resulted in the assay thatwas able to distinguish between clones of higher potency. The protocolfor this modified assay was as described for the originalantibody-ligand biochemical assay using CAN022D10, with the followingchanges. Firstly the concentration of HIS FLAG IL-6 used was reducedfrom 1 nM to 0.5 nM. Secondly, the concentrations of the anti IL-6antibody and streptavidin XLent!™ were increased from 1 nM and 20 nM to16 nM and 40 nM respectively. ScFv that demonstrated a significantinhibitory effect were sequenced and produced as purified scFv and IgG,then tested in the TF-1 proliferation assay.

2.5. Inhibition of IL-6 Induced Proliferation of TF-1 Cells by PurifiedscFv and IgG of Optimised Clones

Potencies of the optimised clones were determined using the IL-6 inducedTF-1 proliferation assay as previously described. Clones were tested asboth purified scFv preparations and as re-formatted IgG. Example resultsfor both scFv and IgG are given in Table 4.

TABLE 4 Example potencies of clones identified from the targetedmutagenesis libraries when tested in the TF-1 cell proliferation assayIC₅₀ (pM) Clone (non-germlined) scFv IgG Antibody 7 11 3 Antibody 8 41948 Antibody 10 549 40 Antibody 14 448 31 Antibody 16 154 4.9 Antibody 1738 16 Antibody 18 51 30 Antibody 19 508 68 Antibody 21 42 N.D. Antibody22 41 N.D. Antibody 23 161 20 CNTO-328 N.D. 74 N.D. Not Determined

Clones demonstrated significant inhibitory effect, but accurate IC₅₀values could not be determined from the dilution series of purifiedscFv.

2.6. Germlining

The amino acid sequences of the VH and VL domains of the optimisedanti-IL-6 antibodies were aligned to the known human germline sequencesin the VBASE database [130], and the closest germline was identified bysequence similarity. For the VH domains of the CANDY022D10 antibodylineage the closest germline v segment was Vh3_DP-86_(3-66) and theclosest germline j segment was JH2. For the VL domains the closestgermline v segment was Vkl_L12 and closest germline j segment was JK2.

Without considering the Vernier residues [131], which were leftunchanged, there were 3 changes in the frameworks of the VH domains and4 changes in the VL domains, all of which were reverted to the closestgermline sequence to identically match human antibodies using standardsite directed mutagenesis techniques with the appropriate mutagenicprimers.

A total of 5 Vernier residues were identified in the scFv sequence ofCAN022D10 that were mutated from germline. These were in the heavy chainat Kabat residues 29 (I present instead of V), 69 (M instead of I), 73(I instead of N) and 78 (V instead of L). A single Vernier mutation wasalso identified in the light chain sequence at Kabat residue 46 (Vinstead of L).

Germlined IgG were then re-evaluated in the IL-6 induced TF-1proliferation assay to confirm there had not been a reduction inpotency. Example potencies for germlined (GL) antibodies are provided inTable 5.

TABLE 5 Example potency data for germlined optimised clones whenevaluated in the IL-6 induced TF-1 cell proliferation assay Clone IC₅₀(pM) Antibody 7 (GL) 5 Antibody 10 (GL) 71 Antibody 17 (GL) 1 Antibody18 (GL) 3 CNTO-328 1012.7. Inhibition of Endogenous IL-6 Induced VEGF Release from Human 5Synovial Fibroblast by Optimised IgG

Optimised IgG were tested in the synovial fibroblast VEGF release assayto evaluate potency against endogenously expressed IL-6. This procedureis reviewed in section 1.7 and described in detail in the Materials andMethods section. Example potencies for the IgG tested are given in Table6a. Mean potency data for the IgG tested are given in Table 6b.

TABLE 6a Example potency data for optimised clones when evaluatedagainst endogenous IL-6 in the IL-6 induced synovial fibroblast VEGFrelease assay Clone (GL = germlined clones) IC₅₀ (nM) Antibody 2 0.59Antibody 3 0.38 Antibody 4 0.52 Antibody 5 0.70 Antibody 7 (GL) 0.75Antibody 10 (GL) 0.55 Antibody 17 (GL) 0.57 Antibody 18 (GL) 0.93CNTO-328 1.31

TABLE 6b Mean potency data for optimised clones when evaluated againstendogenous IL-6 in the IL-6 induced synovial fibroblast VEGF releaseassay Clone (GL = germlined clones) IC₅₀ (nM) (95% CI) n Antibody 7 (GL)0.78 (0.54-1.11) 3 Antibody 17 (GL) 0.57 (0.51-0.64) 3 Antibody 18 (GL)0.67 (0.20-2.25) 4 CNTO-328 1.02 (0.39-2.63) 4

2.8. Selectivity and Species Cross Reactivity of Optimised Antibodies inDELFIA Epitope Competition Assays

Selectivity and species cross reactivity was reevaluated for a panel ofclones using the DELFIA® epitope competition assay as previouslydescribed (see section 1.6 and Materials and Methods). Human andcynomolgus IL-6 produced overlapping inhibition curves and thereforeequivocal IC₅₀ values for all IgG tested. No inhibition was observed formurine, rat or dog IL-6 or any of the related human proteins testedagainst the antibody panel. This data demonstrates that the panel ofclones tested are cross reactive to cynomolgus IL-6 but do not bind tomurine, rat or dog IL-6, or to the most related human proteins to humanIL-6.

2.9 Calculation of Affinity Data for Optimised Clones Using BIAcore

The binding affinity of purified IgG samples of representativeantibodies 7 and 18 to human and cynomolgus IL-6 were determined bysurface plasmon resonance using a BIAcore 2000 biosensor (BIAcore AB)essentially as described in ref. [132]. In brief, purified antibodieswere coupled to the surface of a CM5 sensorchip using an amine couplingkit (BIAcore) to provide a surface density of between 220-225 Ru. Humanand cynomolgus IL-6 at a range of concentrations between 200 nM and 0.2nM in HBS-EP buffer were passed over the sensor chip surface. Theresulting sensorgrams were evaluated using BIA evaluation 3.1 softwareto provide relative binding data.

The lower limit of affinity measurement range of the BIAcore 2000™biosensor is approximately 10 pM (BIAcore 2000 Instrument handbook).From the data obtained, the affinity of the antibodies to both human andcynomolgus IL-6 was below this 10 pM limit, i.e. the antibodies weremore potent than could be measured. Accurate affinity measurements weretherefore not calculated. The affinities of both antibodies to both IL-6species using this approach are considered to be less than 10 pM.

2.10 Calculation of Affinity Data for an Optimised Clone Using the TF-1Cell Proliferation Assay In Vitro

The TF-1 assay was used to calculate the affinity of Antibody 18 by useof Schild analysis. An IL-6 standard curve (7.7×10⁻¹⁵ M to 3×10⁻⁹ M) wasmixed with a range of IgG concentrations (2.67×10⁻¹³ M to 8.3×10⁻¹⁰ M)in duplicate. By plotting the Log 10 antibody concentration against theLog 10 dose ratio, the affinity of the IgG was determined. Using thisapproach the affinity of Antibody 18 (GL) to human IL-6 was calculatedas being 0.40 pM (95% CI 0.12 pM-0.69 pM, n=6).

2.11 Antagonist Potency at Human Recombinant IL-6 Using IL-6 Mediated B9Cell Proliferation In Vitro

IL-6 induced B9 cell proliferation was assessed in the presence ofAntibody 18 and an isotype control antibody. The effects of a range ofconcentrations of each antibody (1×10⁻¹³ M to 1×10⁻⁹ M) were assessed onan IL-6 standard curve (concentration range 1×10⁻¹⁴ M to 1×10⁻⁹ M). Datapoints were in duplicate. B9 proliferation was determined after 4 daysincubation by reduction of alamar blue (fluorescence method).

Antibody 18 was shown to inhibit IL-6 induced B9 proliferation Theisotype control had no inhibitory effect. Mean data are shown in Table8.

TABLE 8 Mean Kb values for inhibition of IL-6 induces B9 proliferationMean Kb pM (95% CI) n Antibody 18 (GL) 0.3 (0.1-0.5) 62.12 Antagonist Potency at Human Recombinant IL-6 Using IL-6 5 MediatedIgM Release from SKW6.4 Cells In Vitro

IL-6 induces IgM secretion from the human B lymphoblast cell line SKW6.4. SKW6.4 cells incubated with a range of IL-6 concentrations (1×10⁻¹³M to 3×10^(−8.5) M) gave an average [A]50 of 77 pM (n=3) on IgMsecretion. The effect of the anti-human IL-6 Antibodies 7, 17 and 18 andan isotype control antibody on IL-6 induced IgM secretion was assessedby observing the inhibition of various antibody concentrations(1×10^(−12.5) M to 1×10⁻⁸ M) in the presence of 100 pM IL-6. IgMsecretion was determined after 4 days by anti-human IgM ELISA. Datapoints were in duplicate.

Antibodies 7, 17 and 18 inhibited IL-6 induced IgM secretion. Theisotype control had no inhibitory effect in these assays. Mean data isshown in Table 9.

TABLE 9 Mean inhibition of IgM secretion from SKW6.4 cells Mean IC50 pMn Antibody 7 (GL) 2.64 3 Antibody 17 (GL) 3.21 3 Antibody 18 (GL) 2.63 3

Example 3. Epitope Mapping 3.1 Comparison of Anti-IL-6 Antibody Epitopeto Known Anti-Human IL-6 Antibodies

The epitope of Antibody 18 (GL) was compared with the epitopes of twoanti-human IL-6 antibodies B-E8 and cCLB. Both these antibodies areknown to inhibit the binding of IL-6 to IL-6Ra and have beeninvestigated as potential therapeutic agents [5, 31, 34, 37, 133]. Toenable comparisons of the epitopes of the three antibodies, a panel ofIL-6 mutants were constructed that each contained a single amino acidmutation compared to the wild-type (wt) sequence. The binding of thesemutants to the different antibodies was then evaluated in biochemicalcompetition assays. These experiments were based on the biochemicalcompetition assay described in Example 1.6, with changes in theconcentrations of antibodies and IL-6 variants where required. Briefly,antibodies were coated on to the surface of a 96-well Nunc Maxisorpimmunoassay plate at a concentration of either 2 nM (Antibody 18) or 4nM (B-E8 and cCLB8) in PBS and incubated overnight at 4° C. After thesurface of the wells was blocked using 3% (w/v) BSA in PBS, dilutions ofthe inhibitors at a concentration range of 200 nM to 10 pM mixed withbiotinylated human IL-6 at a final concentration of 0.15 nM were addedto the antibody coated wells and allowed to bind. Binding of thebiotinylated IL-6 to the antibodies was measured using Europium labelledstreptavidin.

By comparing the IC₅₀ values obtained for the mutants to unlabelled wildtype human IL-6, a ratio of potency could be established for eachmutant. Then, by comparing these ratios across the different antibodies,the effects of the individual mutations on the binding of the antibodyto the IL-6 molecule could be evaluated. Typical results of theseexperiments are presented in Table 10 with the experiments beingrepeated on 2 further occasions.

TABLE 10 IC50 and potency ratios of a panel of IL-6 mutants against theanti-human IL-6 antibodies antibody 18, B-E8 and cCLB8 IC50 (M) PotencyRatio Mutant Antibody 18 CNTO-328 B-E8 Antibody 18 CNTO-328 B-E8 F102E8.41E−08 2.80E−09 1.58E−08 310.951 3.021 57.246 F106E 1.43E−09 Noinhibition 3.31E−09 5.283 — 11.989 Irrelevant No inhibition Noinhibition No inhibition — — — wt IL-6 2.70E−10 9.26E−10 2.76E−10 1.0001.000 1.000 R207E No inhibition 5.13E−09 No inhibition — 7.401 — Q211A3.07E−10 1.10E−07 8.51E−10 1.498 158.221 3.208 Irrelevant No inhibitionNo inhibition No inhibition — — — wt IL-6 2.05E−10 6.93E−10 2.65E−101.000 1.000 1.000 R58E 4.79E−10 1.73E−09 1.57E−08 2.009 1.682 79.083S204E 2.57E−08 1.90E−09 4.83E−10 107.754 1.848 2.434 Irrelevant Noinhibition No inhibition No inhibition — — wt IL-6 2.39E−10 1.03E−091.98E−10 1.000 1.000 1.000 E200W 5.22E−10 2.68E−09 5.68E−10 2.130 3.8172.287 R207L 1.31E−07 1.51E−09 9.41E−08 534.666 2.148 378.865 wt IL-62.45E−10 7.02E−10 2.48E−10 1.000 1.000 1.000

The residue numbering in table 10 is for the amino acid sequence of fulllength human IL-6 (SEQ ID NO: 161).

The results show that the three antibodies have different bindingprofiles against the panel of IL-6 mutants and therefore bind todifferent epitopes on the surface of the cytokine. Kalai et al (1997)previously observed that cCLB8 does not recognise the IL-6 mutant F106E.This has been confirmed in our experiments, as it does not inhibitbinding of the biotinylated IL-6 to the antibody. In contrast, the IL-6mutant F106E is only 5-fold less potent than the wt IL-6 in thecompetition assay using Antibody 18, indicating that it binds stronglyto this antibody. A similar result was observed with mutant Q211A, wherethe potency ratio against antibody 18 was 1.5, compared to 158 forcCLB8. Conversely, mutants F102E, R207E, R207L and S204E were potentinhibitors in the cCLB8 assay but were observed to be considerably lesspotent than wt IL-6 in the Antibody 18 assay.

Differences in the binding of Antibody 18 and B-E8 were observed withmutants R58E and S204E. The potency ratio for R58E was 2.009 forAntibody 18, compared to 79.083 for B-E8, indicating that this mutationreduces the binding of B-E8 to IL-6. The effect of mutation S204Eappears to be specific to Antibody 18 out of the three antibodiestested. As with cCLB8, this mutation has little impact on the potency ofIL-6 binding to B-E8, however the mutant is over 100-fold less potentthan the wild-type IL-6 in the biochemical assay for Antibody 18.

Example 4. Administration of an Anti-IL-6 Antibody In Vivo 4.1 Effect ofAdministration of an Anti-IL-6 Antibody on Human RecombinantIL-6-Induced Neutrophil and Haptoglobin Increase in Mice

Systemic administration of IL-6 is known to cause a systemic increase inneutrophils and acute phase protein concentrations. An in vivo model wasgenerated where human IL-6 was administered by intra-peritonealinjection into male C57/B/6/J mice and concentrations of neutrophils andthe acute phase protein haptoglobin were measured. The ability ofAntibody 18 (GL) administered by sub-cutaneous injection to inhibit theresponses was measured.

4.2 Haptoglobin Assay

Intra-peritoneal injection of human IL-6 (5.2 nmol/kg, equivalent to 12mg/kg, b.i.d.) for 7 days resulted in a significant increase in theplasma haptoglobin levels from 0.02±0.01 mg/mL (vehicle controls) to1.19±0.27 mg/mL in the IL-6 treated group (T-test, P<0.01). Whilst theIgG1 isotype control had no effect, Antibody 18 dose-dependentlyinhibited the response with significant inhibition (ANOVA, P<0.01 vsIL-6 alone) being noted at doses of 10.6 nmol/kg (156 mg/kg) and above(FIG. 1).

4.3 Neutrophil Assay

Intra-peritoneal injection of human IL-6 (5.2 nmol/kg, equivalent to 12mg/kg, b.i.d.) for 7 days resulted in a significant increase inneutrophil count from 1.1±0.44×109 cells/L (vehicle controls) to2.47±0.12×109 cells/L in the IL-6 treated group (T-test, P<0.01). Whilstthe IgG1 isotype control had no effect, antibody 18 dose-dependentlyinhibited the response with significant inhibition (ANOVA, P<0.01 vsIL-6 alone) being noted at doses of 1.5 nmol/kg (23 mg/kg) and above.

These results confirm the ability of an anti-IL-6 antibody to inhibitthe systemic effects of IL-6 in vivo.

Materials and Methods

Inhibition of IL-6 Binding to IL-6 Receptor by Crude scFv

Selection outputs were screened in receptor-ligand binding HTRF®(Homogeneous Time-Resolved Fluorescence) assay format for inhibition ofeither, cryptate labelled human IL-6 (R&D Systems 206-IL), or HIS FLAGtagged human IL-6 (in house E. coli derived) binding biotinylated IL-6R(Peprotech 200-06 R).

Outputs during lead isolation were screened as undiluted, crude scFvcontaining periplasmic extracts prepared in: 200 mM hepes buffer pH7.4,0.5 mM EDTA and 0.5 M sucrose. 8 nM biotinylated human IL-6R waspre-incubated for 30 minutes at room temperature in the dark, with 8 nMstreptavidin XL^(ent!)™ (CIS Bio International 611SAXLA). All dilutionswere done in phosphate buffered saline (PBS) containing 0.4 M potassiumfluoride and 0.1% BSA (assay buffer).

After pre-incubation of the reagents, 10 μl of crude scFv sample wasadded to a 384 well low volume assay plate (Costar 3676). This wasfollowed by the addition of 5 μl of the pre-incubated biotinylatedreceptor and streptavidin XL^(ent!™) mix, and then 5 μl of 11.2 nMcryptate labelled human IL-6.

Assay plates were then centrifuged at 1000 rpm at room temperature for 1min, and incubated for 2 h at room temperature, prior to reading timeresolved fluorescence at 620 nm and 665 nm emission wavelengths using anEnVision plate reader (Perkin Elmer).

Inhibition of IL-6 Binding to IL-6 Receptor by Purified scFv and IgG

Purified scFv and IgG from positive clones identified from screeningwere tested in a HTRF® assay for inhibition of binding of HIS FLAGtagged human IL-6 to biotinylated IL-6R. 8 nM biotinylated human IL-6Rwas pre-incubated for 30 minutes at room temperature in the dark, with 8nM streptavidin XL^(ent!)™. All dilutions were done in phosphatebuffered saline (PBS) containing 0.4 M potassium fluoride and 0.1% BSA(assay buffer).

A titration of the purified sample was used in order to establish theclone potency as measured by IC₅₀ values in the assay. Afterpre-incubation of the reagents, 10 μl of titration of purified scFvsample was added to a 384 well low volume assay plate (Costar 3676).This was followed by the addition of 5 μl of the pre-incubatedbiotinylated receptor and streptavidin XL^(ent!™) mix. 2 nM HIS FLAGtagged human IL-6 was combined with 1.732 nM anti-flag IgG labelled withcryptate (CIS Bio International 61FG2KLB) and immediately 5 μl of mixwas added to assay plate.

Assay plates were then centrifuged at 1000 rpm at room temperature for 1min, and incubated for 2 h at room temperature, prior to reading timeresolved fluorescence at 620 nm and 665 nm emission wavelengths using anEnVision plate reader (Perkin Elmer).

Data Analysis

The following methods were used to analyse data from the HTRF® assaysdescribed above.

Data was analysed by calculating % Delta F values for each sample. DeltaF was determined according to equation 1.

$\begin{matrix}{{\% \mspace{14mu} {Delta}\mspace{14mu} F} = {\frac{\begin{matrix}{\left( {{sample}\mspace{14mu} 665\mspace{14mu} {{nm}/620}\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right) -} \\\left( {{non}\text{-}{specific}\mspace{14mu} {control}\mspace{14mu} 665\mspace{14mu} {{nm}/620}\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right)\end{matrix}}{\left( {{non}\text{-}{specific}\mspace{14mu} {control}\mspace{14mu} 665\mspace{14mu} {{nm}/620}\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right)} \times 100}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

% Delta F values were subsequently used to calculate % specific bindingas described in equation 2.

$\begin{matrix}{{\% \mspace{14mu} {specific}\mspace{14mu} {binding}} = {\frac{\% \mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} {of}\mspace{14mu} {sample}}{\% \mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} {of}\mspace{14mu} {total}\mspace{14mu} {binding}\mspace{14mu} {control}} \times 100}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

IC₅₀ values were determined using GraphPad Prism software by curvefitting using a four-parameter logistic equation (Equation 3).

Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((LogEC50−X)*HillSlope))  Equation 3:

X is the logarithm of concentration. Y is specific binding

Y starts at Bottom and goes to Top with a sigmoid shape.

A reference anti-IL-6 mAb (Biosource AHC0562) was included in all assaysas a positive control.

Inhibition of IL-6 Induced Proliferation of TF-1 Cells by Purified scFvand IgG

TF-1 cells were a gift from R&D Systems and maintained according tosupplied protocols. Assay media comprised RPMI-1640 with GLUTAMAX I(Invitrogen) containing 5% foetal bovine serum (JRH) and 1% sodiumpyruvate (Sigma). Prior to each assay, TF-1 cells were pelleted bycentrifugation at 300×g for 5 mins, the media removed by aspiration andthe cells re-suspended in assay media. This process was repeated twicewith cells re-suspended at a final concentration of 5×10⁵ cells/ml inassay media. The cells were plated out using 100 μl/well in a 96 wellassay plate. Plates were incubated for 24 hours at 37° C. and 5% CO₂ tostarve cell of GM-CSF. Test solutions of purified scFv or IgG (induplicate) were diluted to the desired concentration in assay media. Anirrelevant antibody not directed at IL-6 was used as negative control.Recombinant bacterially derived human (R&D) and cynomolgus (in-house)IL-6 was added to a final concentration of either 20 pM (human IL-6) or100 pM (cynomolgus) when mixed with appropriate test antibody in a totalvolume of 100 μl/well. The concentration of IL-6 used in the assay wasselected as the dose that at final assay concentration gaveapproximately 80% of maximal proliferative response. All samples wereincubated for 30 mins at room temperature. 100 μl of IL-6 and antibodymixture was then added to 100 μl of the cells to give a total assayvolume of 200 μl/well. Plates were incubated for 24 hours at 37° C. and5% CO₂, 20 μl of tritiated thymidine (5 μCi/ml) was then added to eachassay point and the plates were returned to the incubator for further 24hours. Cells were harvested on glass fibre filter plates (Perkin Elmer)using a cell harvester. Thymidine incorporation was determined usingPackard TopCount microplate liquid scintillation counter. Data was thenanalysed using Graphpad Prism software.

Method for Time Resolved Fluorescence Assay of Inhibition ofBiotinylated Human IL-6 Binding to Immobilised Anti IL-6 Antibodies

The specific method used for this assay and for which results areprovided in Example 2.6 employed DELFIA® reagents and is set out above.The method is also described more generally below, and is suitable as anassay for determining and/or quantifying binding of other IL-6 forms andrelated proteins to anti IL-6 MAbs.

In this assay, the anti-IL-6 monoclonal antibody is bound to a solidsupport, for example being attached to the support via Fc. Polystyrenehigh protein binding plates, e.g. Nunc Maxisorb plates, may be used as asuitable support.

-   -   Coat the anti IL-6 MAb on to plates at 50 μl per well in PBS,        overnight at 4° C.    -   All subsequent steps are performed at room temperature.    -   Wash plates three times with PBS, containing 0.05% Tween20        (PBST, currently available under Sigma P1379), then block with        300 μl/well PBS containing 3% (w/v) BSA (currently available        under Roche Diagnostics, 70129138) for 1 h.    -   Wash plates three times with PB ST.    -   Prepare inhibitor titrations in PBS containing 3% (w/v) BSA and        add to a ‘dilution’ plate (40 μl/well) followed by 40 μl/well        biotinylated IL-6 to give a final concentration of biotinylated        IL-6 equivalent to the KD for the protein for the antibody.        Transfer 50 μl of the samples from the dilution plate to the        corresponding wells in the assay plate    -   Incubate plates for 1 h.    -   Wash plates three times with PEST then to each well add 50        μl/well of 0.1 μg/ml Europium-labelled streptavidin in 50 mM        Tris-HCl, pH 7.5, containing 0.9% NaCl, 0.5% purified BSA, 0.1%        Tween20 and 20 m EDTA and incubate for 1 h.    -   Wash plates seven times with a wash buffer comprising of 0.05M        Tris buffered saline (0.138M NaCl, 0.0027M KCl), 0.05% (v/v)        Tween20, pH8.0 (at 25° C.)    -   To each well, add 50 μl of an enhancement solution, acidified        with acetic acid and containing Triton X-100 along with the        chelators βNTA and TOPO. The resulting pH shift from alkali to        acid causes a rapid dissociation of the europium ions from the        streptavidin conjugate. The free Europium ions then form        fluorogenic chelates with the available chelators. Water is        removed by the presence of TOPO, enabling the chelates to form        micelles, prolonging the fluorogenicity of the chelate.    -   Incubate for 5 min, then measure time resolved fluorescence at a        620 nm emission wavelength. Fluorescence data are converted to %        specific binding according to Equation 1. Determine total        binding from control wells containing biotinylated huIL-6 but no        competitor. Determine non-specific binding from wells containing        biotinylated huIL-6 and a 100-fold excess of huIL-6. Fit        resultant data to a sigmoidal curve for calculation of IC₅₀        values according to Equation 2.        Determination of Antibody Coating and Biotinylated huIL-6        Concentrations for the Biochemical Epitope Competition Assay

The concentration of antibody used for coating and the concentration ofbiotinylated huIL-6 used in the epitope competition assay will depend onthe affinity of the interaction of the two reagents and the efficiencyof antibody immobilisation. A standard concentration for antibodycoating and the concentration of biotinylated huIL-6 required musttherefore be determined for each antibody to be tested.

As a general rule, the final concentration biotinylated huIL-6 used ineach assay is equlivalent to the KD of the ligand for the correspondingantibody as determined by saturation analysis. The concentration ofantibody used for coating should be such that when the biotinylatedhuIL-6 is added at KD a minimum signal to background ratio of 10:1 isobtained when detected under the competition assay conditions.

Selectivity and Species Cross Reactivity of Antibodies in DELFIA®Epitope Competition Assays

Purified IgG were adsorbed onto 96-well Maxisorp microtitre plates(Nunc) in PBS at a concentration which gave a significant signal whenbiotinylated human IL-6 was added at approximately its estimated Kd forthat particular IgG. Excess IgG was washed away with PBS-Tween (0.1%v/v) and the wells were blocked with PBS-Marvel (3% w/v) for 1 h. Adilution series of each of the following competitors was prepared inPBS, starting at a concentration of approximately 200-times the Kd valueof the Interaction between biotinylated human IL-6 and the respectiveIgG; Human IL-6, Cynomolgus IL-6, Rat IL-6 (R & D Systems 506-RL/CF),Murine IL-6 (R & D Systems 406-ML/CF), Human CNTF (R & D Systems257-NT/CF), Human LIF (Chemicon, LIF 1010), Human IL-11(R & D Systems518-IL/CF) Human Oncostatin M (R & D Systems 295-OMICF) Unbiotinylatedhuman IL-6 was used as a positive control. To this series, an equalvolume of biotinylated recombinant human IL-6 at a concentration ofapproximately 2-fold the Kd was added (resulting in a series starting ata ratio of competitor antigen:biotinylated human IL-6 of approximately100:1). These mixtures were then transferred onto the blocked IgG andallowed to equilibrate for 1.5 h. Unbound antigen was removed by washingwith PBS-Tween (0.1% v/v), while the remaining biotinylated human IL-6was detected by streptavidin-Europium3+ conjugate (DELFIA®, detection,PerkinElmer). Time-resolved fluorescence was measured at 620 nm on anEnVision plate reader (PerkinElmer). Fluorescence data was converted to% specific binding (100% was determined from control wells containingbiotinylated human IL-6 but no competitor, 0% was from wells containingbiotinylated human IL-6 and a 100-fold excess of unbiotinylated humanIL-6). Resultant data were analysed using Prism curve fitting software(Graphpad) to determine IC50 values according to Equation 3.

Method for Time Resolved Fluorescence Assay of Inhibition ofBiotinylated Human IL-6 Binding to Immobilised Anti IL-6 Antibodies

The specific method used for this assay and for which results areprovided in Example 2.8 employed DELFIAC, reagents and is set out above.The method is also described more generally below, and is suitable as anassay for determining and/or quantifying binding of other IL-6 forms andrelated proteins to anti IL-6 MAbs.

In this assay, the anti-IL-6 monoclonal antibody is bound to a solidsupport, for example being attached to the support via Fc. Polystyrenehigh protein binding plates, e.g. Nunc Maxisorb plates, may be used as asuitable support.

-   -   Coat the anti IL-6 MAb on to plates at 50 μl per well in PBS,        overnight at 4° C.    -   All subsequent steps are performed at room temperature.    -   Wash plates three times with PBS, containing 0.05% Tween20        (PBST, currently available under Sigma P1379), then block with        300 μl/well PBS containing 3% (w/v) BSA (currently available        under Roche Diagnostics, 70129138) for 1 h.    -   Wash plates three times with PB ST.    -   Prepare inhibitor titrations in PBS containing 3% (w/v) BSA and        add to a ‘dilution’ plate (40 μl/well) followed by 40 μl/well        biotinylated IL-6 to give a final concentration of biotinylated        IL-6 equivalent to the KD for the protein for the antibody.    -   Transfer 50 μl of the samples from the dilution plate to the        corresponding wells in the assay plate    -   Incubate plates for 1 h.    -   Wash plates three times with PBST then to each well add 50 t        1/well of 0.1 g/ml Europium-labelled streptavidin in 50 mM        Tris-HCl, pH 7.5, containing 0.9% NaCl, 0.5% purified BSA, 0.1%        Tween20 and 20 m EDTA and incubate for 1 h.    -   Wash plates seven times with a wash buffer comprising of 0.05M        Tris buffered saline (0.138M NaCl, 0.0027M KCl), 0.05% (v/v)        Tween20, pH8.0 (at 25° C.)    -   To each well, add 50 μl of an enhancement solution, acidified        with acetic acid and containing Triton X-100 along with the        chelators (3NTA and TOPO. The resulting pH shift from alkali to        acid causes a rapid dissociation of the europium ions from the        streptavidin conjugate. The free Europium ions then form        fluorogenic chelates with the available chelators. Water is        removed by the presence of TOPO, enabling the chelates to form        micelles, prolonging the fluorogenicity of the chelate.    -   Incubate for 5 min, then measure time resolved fluorescence at a        620 nm emission wavelength. Fluorescence data are converted to %        specific binding according to Equation 1. Determine total        binding from control wells containing biotinylated huIL-6 but no        competitor. Determine non-specific binding from wells containing        biotinylated huIL-6 and a 100-fold excess of huIL-6. Fit        resultant data to a sigmoidal curve for calculation of IC50        values according to Equation 2.        Determination of Antibody Coating and Biotinylated huIL-6        Concentrations for the Biochemical Epitope Competition Assay

The concentration of antibody used for coating and the concentration ofbiotinylated huIL-6 used in the epitope competition assay will depend onthe affinity of the interaction of the two reagents and the efficiencyof antibody immobilisation. A standard concentration for antibodycoating and the concentration of biotinylated huIL-6 required musttherefore be determined for each antibody to be tested.

As a general rule, the final concentration biotinylated huIL-6 5 used ineach assay is equlivalent to the KD of the ligand for the correspondingantibody as determined by saturation analysis. The concentration ofantibody used for coating should be such that when the biotinylatedhuIL-6 is added at KD a minimum signal to background ratio of 10:1 isobtained when detected under the competition assay conditions.

Identification of Improved Clones Using an Antibody-Ligand BiochemicalAssay

Selection outputs from lead optimisation were screened in epitopecompetition HTRF® assay format for inhibition of HIS FLAG tagged humanIL-6 (in house E. coli derived) binding biotinylated anti IL-6 antibody(in house IgG derived from lead isolation, CAN022D10).

Outputs during lead optimisation were screened as undiluted, crude scFvcontaining periplasmic extracts prepared in; 50 nM MOPS buffer pH7.4,0.5 mM EDTA and 0.5M Sorbitol. 1 nM human HIS FLAG IL-6 waspre-incubated for 30 minutes at room temperature in the dark, with 1.732nM anti-flag IgG labelled with cryptate (CIS Bio International61FG2KLB). All dilutions were performed in assay buffer. In parallel, 1nM of biotinylated anti-IL-6 IgG (against which competition of a testbinding member was to be tested) was pre-incubated for 30 minutes atroom temperature in the dark with 20 nM of streptavidin XL^(ent!)™ (CISBio International 611SAXLB).

After pre-incubation of reagents, 10 pl of crude scFv sample was addedto a black 384 well optiplate (Perkin Elmer Cat No. 6007279). This wasfollowed by addition of 10 μl assay buffer to the whole plate. Then 10μl of the pre-incubated biotinylated anti-IL-6 IgG and StreptavidinXLentl™ mix, and 10 μl of pre-incubated HIS FLAG tagged human IL-6anti-flag cryptate mix were added.

Assay plates were then centrifuged at 1000 rpm at room temperature for 1min, and incubated for 2 h at room temperature, prior to reading timeresolved fluorescence at 620 nm and 665 nm emission wavelengths using anEnVision plate reader (Perkin Elmer). Data was analysed by calculating %deltaF and % specific binding as previously described.

Following identification of improved leads from the random mutagenesislibrary, undiluted crude scFv outputs from CDR3 targeted mutagenesisselections were screened in a modified version of the epitopecompetition HTRF® assay which included the following changes 0.5 nMhuman HIS FLAG IL-6 was pre-incubated for 30 minutes at room temperaturein the dark, with 1.732 nM anti-flag IgG labelled with cryptate (CIS BioInternational 61FG2KLB). In parallel, 16 nM of biotinylated anti-IL-6IgG (Antibody 5, in house IgG identified from CAN022D10 randommutagenesis selections) was pre-incubated for 30 minutes at roomtemperature in the dark with 40 nM of streptavidin XL^(ent!)™ (CIS BioInternational 611SAXLB).

All other conditions were as described for CAN022D10 epitope competitionassay. Data were analysed by calculating % deltaF and % specific bindingas previously described.

Inhibition of Endogenous IL-6 Induced VEGF Release from Human SynovialFibroblasts by Purified IgG

Samples of rheumatoid arthritis knees from total joint replacementsurgery were obtained in DMEM containing antibiotics. Synovium bathed inmedia was dissected from the joint & finely chopped. The synovial tissuewas washed with media supplemented with 10% FCS. The cell suspension wasincubated in a collagenase solution for 2 hours in a CO₂ incubator at37° C. The digested synovial cell suspension was disrupted by repeatedlyaspirating through a 10 ml pipette, cell strained & centrifuged at 400 gat room temperature for 5 minutes. The cells were resuspended in DMEMcontaining 10% FCS, passed through a cell strainer, adjusted to 1×10⁶cells per ml & incubated in a CO₂ incubator at 37° C. in 225-cm² cellculture flasks (3001, CoStar Corning Inc.). Following adherence, themajority of the medium was discarded, replaced with fresh & returned tothe incubator for long-term incubation. The cells were examined on aweekly-basis & were passaged at confluence by trypsinisation at apassage rate of 1 in 3.

Fibroblasts (P3-5) at confluence were removed from flasks by incubatingwith 10 mL 0.1% trypsin-EDTA solution (25300-054, Gibco Life Sciences)per flask for 5 to 10 minutes at 37° C. An equal volume of DMEM-basedculture medium supplemented with 10% FCS was added to the cells, whichwere then pelleted by centrifugation at 330 g for 5 minutes at RT. Afterone wash step with DMEM-based culture medium supplemented with 10% FCS,the cell suspension (1×10⁵ cells per mL) was added (150 pL per well) towells of sterile 96 well cell culture cluster flat bottom polystyreneplates (3598, Corning CoStar) at 1.5×10⁴ cells per well. A furtheraddition of DMEM-based culture media supplemented with 10% FCS was addedto each well (100 μL per well) to give a total volume of 250 μL perwell. The cells were incubated at 37° C. overnight to allow foradherence and quiescence.

The 96-well plates were inspected to ensure that the cells wereconfluent and in good condition (e.g. contamination-free). Medium wasthen aspirated from the wells and 100 μL of DMEM-based culture mediumsupplemented with 10% FCS was immediately added. To this, 50 μL ofDMEM-based culture medium supplemented with 10% FCS containing eithersample IgG or medium alone was added to the wells (diluted 1 in 5 intoassay).

This was followed by adding 50 μL per well of DMEM-based culture mediumsupplemented with 10% FCS containing recombinant human soluble (rhs)IL-6Rα (500 ng per mL; 12 nM) and rhIL-1β (50 pg per mL; 2.95 pM,diluted 1 in 5 into assay).

In separate wells, 50 μL of DMEM-based culture medium supplemented with10% FCS containing either; rh-IL-6 (0, 100 ng per mL; 21.5 nM), sIL-6Rα(500 ng per mL; 12 nM), rhIL-1β (50 pg per mL; 2.95 pM), or medium alonewas added (diluted 1 in 5 into assay). Final volume in each well was 250μL.

The plates were incubated for 48 hours at 37° C. Incubations wereperformed in duplicate or triplicate wells as described in the plateformat. The plates were centrifuged at 330 g for 5 minutes at RT andsupernatant media was removed and stored at −40° C. in microtitre flatbottom plates (611F96, Sterilin).

VEGF was measured using an ELISA (DY293B, R&D Systems) following themanufacturers instructions. Briefly, ELISA plates were coated with amouse anti-human VEGF antibody overnight at 4° C. and blocked with 1%BSA/PBS. Plates were washed with 0.05% Tween 20/PB S and incubated withculture supernatants of human synovial derived fibroblasts and abiotinylated goat anti-human VEGF antibody over night at roomtemperature. After washing, VEGF was detected by using Streptavidinhorseradish peroxidase. Plates were developed using 1:1H₂O₂:tetramethylbenzidine. The reaction was stopped with 2 M H₂SO₄, andoptical densities were determined at 450 nm with the correctionwavelength set at 540 nm.

BIAcore Measurements

BIAcore studies were undertaken using a BIAcore 2000™. Antibodies werecoupled to the surface of a CM-5 sensorchip using an amine coupling kitto provide a surface density of 220-225 Ru. Human IL-6 at a range ofconcentrations between 200 nM and 0.2 nM in HBS-EP buffer were passedover the sensor chip surface. The resulting sensorgrams were evaluatedusing BIA evaluation 3.1 software to calculate the k_(on), k_(off) andK_(D) values for the antibodies tested.

IL-6 Mediated B9 Cell Proliferation Assay

B9 cells are a sub-clone of the murine B-cell hybridoma cell line,B13.29, selected on the basis of their specific response to IL-6.

B9 cells require IL-6 for survival and proliferation and respond to verylow concentrations of IL-6.

IL-6 induced B9 cell proliferation was assessed in the presence ofAntibody 18 and an isotype control (CAT-002). The effects of a range ofconcentrations of each antibody (1×10⁻¹³ M to 1×10⁻⁹ M) were assessed onan IL-6 standard curve (concentration range 1×10⁻¹⁴ M to 1×10⁻⁹ M) Datapoints were in duplicate. B9 proliferation was determined after 4 daysincubation by reduction of alamar blue (fluorescence method).

B9 cells were cultured in RPMI-1640 containing 5% FCS, 2 mM L-Glutamineand 50 μM 2-mercaptoethanol. Cells were split every 2 to 4 days to adensity of between 0.05×10⁶ mL⁻¹ and 0.1×10⁶ mL⁻¹ and supplemented with5×10 M human IL-6. Cells used for experiments were not supplemented withIL-6 for at least 48 hours prior to experiment but had been supplementedwithin 96 hours of experiment. Cells used in the assay were taken from astock flask with a density of no greater than 0.8×10⁶ mL⁻¹.

Each antibody was diluted from stock solutions to 10× the maximumrequired assay concentration by appropriate dilutions in assay media(RPMI+5% FCS, 2 mM L-Glutamine, 50 μM 2-mercaptoethanol, penicillin 100UmL⁻¹ and streptomycin 100 mgmL⁻¹). Further 10 fold dilutions in culturemedia were carried out to obtain the required concentrations of eachantibody.

IL-6 was reconstituted from a lyophilised powder to a 1×10⁻⁵ M solutionby addition of an appropriate volume of sterile PBS+0.1% BSA. A furtherdilution to 1×10⁻⁸ M was carried out in culture media. 1×10⁻⁸ M aliquotswere stored frozen until required. On the day of assay 1×10⁻⁸ M aliquotswere diluted as necessary to achieve the range of solutions at 10× finalassay concentration required.

The required volume of cells was removed from culture flasks andcentrifuged at 300 g for 8 minutes. Supernatants were removed and thecells re-suspended in an appropriate volume of culture media to achievea cell density of 0.5×10⁶ mL⁻¹.

Assays were performed in flat-bottomed, tissue culture treated,polystyrene 96 well plates. The final assay volume was 200 μL. 20 μL of10× antibody (Antibody 18 or CAT-002) solution or culture media wasadded to the appropriate wells of each plate followed by a further 140μL of culture media and 20 μL of the appropriate concentration of IL-6or culture media.

Plates were placed in a humidified 5% CO₂, 37° C. incubator for 2 hours.20 μL of cells was then added to each well. Final number of cells perwell was 10000. Plates were then returned to the incubator for 4 days.Cell proliferation was assessed by incorporation of alamar blue. 10% v/valamar blue was added to each well and the plates returned to theincubator for 6 hours. Plates were then read on a spectrofluorimetermeasuring fluorescence at 590 nm following excitation at 544 nm Raw datawere normalised to the control IL-6 curve on the each plate such thatmaximum fluorescence was defined as 100% and the basal fluorescence 0%.Normalised data was fitted using the non-linear regression, sigmoidaldose-response (variable slope) fitting programme in Graph Pad Prism4.01. Control pEC₅₀ values and pEC₅₀ values in the presence of eachconcentration of antibody were used to determine dose ratios (DR). Kbvalues were determined for the lowest concentration of antibody whichelicited a 3-fold or greater shift in the IL-6 concentration-effectcurve using the chemical antagonism equation below:

K _(b)=([Ab]/(DR−1))

-   (Kenakin T P, In: Pharmacologic Analysis of Drug-Receptor    Interactions. 1st ed. New York: Raven Press; 1987. p. 205-24)

IL-6 Mediated SKW6.4 Cell IgM Release Assay

IL-6 is involved in the final maturation of B cells into antibodyproducing cells (B-lymphocyte differentiation). SKW cells have been usedpreviously for the study of B cell responses (Nawata et al., Ann. N.Y.Acad. Sci. 557:230-238. 1989). Auto-antibody production in rheumatoidarthritis is mostly of the IgM class. SKW6.4 is a clonal IgM secretinghuman lymphoblastoid B cell line. Cells were sourced from ATCC,reference # TIB 215. Upon stimulation with IL-6 these cells secrete IgM,thus this assay was perceived to be relevant to rheumatoid arthritis.

IL-6 induced SKW6.4 cell IgM secretion was assessed in the presence ofCAT6001 and CAT-002 (isotype control). The effects of a range ofconcentrations of each antibody (1×10^(−12.5) M to 1×10⁻⁸ M) wereassessed in the presence of 100 pM IL-6. Data points were in duplicate.IgM secretionin the cell supernatants was determined after 4 daysincubation using anti-human IgM ELISA assay.

SKW 6.4 cells were cultured in RPMI1640 containing 2 mM L-Glutamine and10% (v/v) foetal calf serum at 37° C. at 95/5% (v/v) air/CO₂ in 95%relative humidity. The cells were maintained between 0.4 and 2×10⁶cells/ml. For routine cell passage, cells were harvested bycentrifugation at 300×g for 5 minutes at room temperature, spent mediumwas removed and the cells re-suspended in the required volume of freshmedia.

Each antibody was diluted from stock solutions to 50× the maximumrequired assay concentration by appropriate dilutions in assay media(RPMI+10% FCS, 2 mM L-Glutamine). Further 10 fold dilutions in culturemedia were carried out to obtain the required concentrations of eachantibody.

Assays were performed in flat-bottomed, tissue culture treated,polystyrene 96 well plates. SKW 6.4 cell stocks were diluted to a celldensity of 0.3×10⁶ ml⁻¹ in fresh media, and plated at 100 μl/well,(30,000 cells per well). 2 μl of antibody, at the indicated finalconcentration, followed by 2 μl of IL-6 at a final concentration of 100pM was then added to each well.

Plates were then returned to the incubator at 37° C. 5% CO₂. Cell-freesupernatants were harvested after 4 days incubation by centrifugationand then either assayed by IgM ELISA on the day of harvest or frozen at−20° C. prior to further analysis.

An ELISA was generated using a pair of antibodies from Serotec. Thecoating antibody was Mouse anti-human IgM (MCA1662) and the detectionantibody was Goat anti-human IgM: HRP linked (STAR98P). The assay wasoptimised by standard methods to give a good signal to noise ratio usingcoating antibody @ 1:2000 dilution (5 g/ml) and detection antibody @1:3500 dilution (200 ng/ml).

IgM standard solution (Cat # PHP003 Human M Kappa purified protein) waspurchased from Serotec to generate a standard curve.

Data was analysed using a polynomial fit for the IgM standard curve datausing a standard fitting programme. The percentage inhibition of eachantibody sample against the control IgM production in the absence ofantibody was calculated and IC₅₀ values were generated.

Generation of IL-6 and IL-6 Mutant Proteins for Epitope Mapping

Cloning of Human and Cyno IL-6 cDNA

The sequences of human and macaque IL-6 were obtained from Embl(Accession No: BC015511 and AB000554 for human and cyno, respectively).Using these sequences oligonucleotide primers were designed to amplifythe cDNA encoding human & macaque IL-6. The N-terminal primers werehIL6-5′NdeI and macIL6 5′ NdeI for human and cyno respectively andmacTL6 3′NheI was used as the C terminal primer for both (See Table 11for oligonucleotides sequences).

TABLE 11 Primer sequences Primer Sequence macIL6_5′NdeI 5′TTATCAT-ATGGTACTCCCAGGAGAAGATT CCAA 3′ (SEQ ID NO: 183) macIL6_3′NheI 5′TTATGCTAGC-CTACATTTGCCGAAGAGCC C 3′ (SEQ ID NO: 184) hIL6_5′NdeI 5′TTATACATATG-GTACCCCCAGGAGAAGAT TCC 3′ (SEQ ID NO: 185)

PCVR reactions to amplify the two cDNAs were carried out. The templatefor each PCR reaction was 10 ng of cDNA obtained from human Liver andcynomolgus liver respectively. The amplified cDNA from each reaction waspurified and cloned into pCR4blunt topo (Invitrogen) using thetopoisomerase ligation reaction according to the manufacturer.

Positive clones were identified and sequenced. The resulting cDNAs 10were sub-cloned using standard techniques into various E. coliT7-promoter expression vectors in such a way that the cDNA encodingmature human or cynomolgus IL-6 were fused at the N-terminus with eitheran N-terminal HIS6-FLAG tag immediately upstream of the N-terminalvaline of mature IL-6.

Generating Mutants

Site directed mutagenesis was performed using a Quikchange XL kit fromStratagene according to the manufacturer's protocol. Mutagenesis primerdesign was performed according to the manufacturer's protocol.Mutagenesis reactions were carried out according to the protocol usingplasmid pT7flagHISIL-6 as template. This was followed by subsequent DpnIdigestion and transformation into chemically competent Top 10 cells withselection on agar plates containing appropriate antibiotics at 37° C.overnight. For each individual mutagenesis reaction several clones weresequenced and plasmid DNA of one correct clone from each reaction wasretained for further use.

Expression of IL-6 and IL-6 Mutant Proteins

The IL-6 expression plasmids were transformed into chemically competentBL21 (DE3) star cells (Invitrogen) using the manufacturer's method.Transformed cells were used to inoculate 1L cultures of Terrific Brothand these were incubated on an orbital incubator at 37° C., until theA600 reached 0.5. IPTG was then added to 0.25 mM and incubationcontinued overnight at 22° C. The cells were harvested by centrifugationand the cell pellets were stored at −80° C.

Purification of IL-6 and IL-6 Mutant Proteins

The cell pellets were thawed and resuspended in 50 ml per pellet of 50mM potassium phosphate, pH7.4, 10 mM imidazole, 0.3M NaC 1, 5 mMbeta-mercaptoethanol, 10% glycerol (buffer A)+Complete EDTA-freeprotease inhibitors (Roche). The cells were lysed by sonication for 3×30seconds on ice. The lysate was centrifuged at 100,000 g and 4° C. for 30minutes and the supernatant was subjected to Ni NTA affinitychromatography. A 5 ml column of Ni-NTA Superflow (Qiagen) wasequilibrated at 3 ml/min with (buffer A). The IL-6 sample was loaded andthe column was washed with 10 column volumes of 15 mM imidazole inbuffer A. This was followed by a 10 column volume wash with 30 mMimidazole in buffer A. IL-6 was eluted from the column using a 5 columnvolume wash in the upward flow direction with 0.3M imidazole in bufferA. 10 ml fractions were collected during the wash steps and 5 mlfractions were collected during the elution step. The column was run at4° C. using the AKTA Explorer100 Air. Fractions containing the purifiedIL-6 protein were pooled and dialysed overnight at 4° C. against 5 L ofPBS.

The dialysed IL-6 proteins were further purified using gel filtrationchromatography. For each purification the dialysed IL-6 protein wascentrifuged at 100,000 g and 4° C. for 20 minutes. Up to 13 ml wasapplied to a 318 ml Superdex 200 26/60 column (GE Healthcare) that hadbeen equilibrated in PBS at 2.5 ml/min. The column was run at 4° C.using an AKTA Purifier. Fractions containing the monomeric IL-6 proteinpeak were pooled for further analysis.

Each protein was checked for purity using standard SDS-chromatography,the protein concentration was measured and Q-ToF mass spectroscopy wasused to measure the mass of the protein. Purified IL-6 was frozen inliquid nitrogen and stored at −80° C.

Materials and Methods for In Vivo Studies

Animals were randomly assigned to into test groups. The mice in eachtest group were then treated daily with set sub-cutaneous doses (10ml/kg) of either vehicle control (0.05% BSA in PBS) or 467 μg/kg IgG1isotype control or antibody 18 (range from 467 μg/kg to 8 μg/kg). At thesame time the mice were given an intra-peritoneal injection (10 ml/kg)b.i.d. of either vehicle control (0.05% BSA in PBS) or 12 g/kg humanrecombinant IL-6.

On day 7, two hours following the final IL-6 dose at 09:00 h, the micewere sacrificed and terminal blood samples were taken. The blood wastransferred to Lab Tek lml EDTA blood tubes, which were placed on aroller for 5 minutes. Samples were then kept on ice until used.Differential cell counts were performed using a Sysmex cell counter. Theremainder of the sample was transferred to an eppendorf tube and spun(300 g, 5 mins) to obtain plasma which was sub aliquoted and stored at−20° C. until anlaysed for Haptoglobin levels.

The haptoglobin assay was carried out as per instructions provided inthe PHASE™ RANGE TriDelta Format kit by Biognosis (Hailsham, UK; cat.no. TP-801).

All results were expressed as mean+ SEM. Data analysis was by unpairedT-test or one-way ANOVA followed by Dunnett's test (GraphPad Instat).

Sequences

VH domain, VL domain and CDR sequences of binding members are shown inthe appended sequence listing, in which SEQ ID NOS correspond asfollows:

1 CAN022D10 VH nucleotide 2 CAN022D10 VH amino acid 3 CAN022D10 VH CDR 1aa 4 CAN022D10 VH CDR 2 aa 5 CAN022D10 VH CDR 3 aa 6 CAN022D10 VLnucleotide 7 CAN022D10 VL amino acid 8 CAN022D10 VL CDR 1 aa 9 CAN022D10VL CDR 2 aa 10 CAN022D10 VL CDR 3 aa 11 Antibody 2 VH nucleotide 12 Ab 2VH amino acid 13 Ab 2 VH CDR 1 amino acid 14 Ab 2 VH CDR 2 amino acid 15Ab 2 VH CDR 3 amino acid 16 Ab 2 VL nucleotide 17 Ab 2 VL amino acid 18Ab 2 VL CDR 1 amino acid 19 Ab 2 VL CDR 2 amino acid 20 Ab 2 VL CDR 3amino acid 21 Antibody 3 VH nucleotide 22 Ab 3 VH amino acid 23 Ab 3 VHCDR 1 amino acid 24 Ab 3 VH CDR 2 amino acid 25 Ab 3 VH CDR 3 amino acid26 Ab 3 VL nucleotide 27 Ab 3 VL amino acid 28 Ab 3 VL CDR 1 amino acid29 Ab 3 VL CDR 2 amino acid 30 Ab 3 VL CDR 3 amino acid 31 Antibody 4 VHnucleotide 32 Ab 4 VH amino acid 33 Ab 4 VH CDR 1 amino acid 34 Ab 4 VHCDR 2 amino acid 35 Ab 4 VH CDR 3 amino acid 36 Ab 4 VL nucleotide 37 Ab4 VL amino acid 38 Ab 4 VL CDR 1 amino acid 39 Ab 4 VL CDR 2 amino acid40 Ab 4 VL CDR 3 amino acid 41 Antibody 5 VH nucleotide 42 Ab 5 VH aminoacid 43 Ab 5 VH CDR 1 amino acid 44 Ab 5 VH CDR 2 amino acid 45 Ab 5 VHCDR 3 amino acid 46 Ab 5 VL nucleotide 47 Ab 5 VL amino acid 48 Ab 5 VLCDR 1 amino acid 49 Ab 5 VL CDR 2 amino acid 50 Ab 5 VL CDR 3 amino acid51 Antibody 7 VH nucleotide 52 Ab 7 VH amino acid 53 Ab 7 VH CDR 1 aminoacid 54 Ab 7 VH CDR 2 amino acid 55 Ab 7 VH CDR 3 amino acid 56 Ab 7 VLnucleotide 57 Ab 7 VL amino acid 58 Ab 7 VL CDR 1 amino acid 59 Ab 7 VLCDR 2 amino acid 60 Ab 7 VL CDR 3 amino acid 61 Antibody 8 VH nucleotide62 Ab 8 VH amino acid 63 Ab 8 VH CDR 1 amino acid 64 Ab 8 VH CDR 2 aminoacid 65 Ab 8 VH CDR 3 amino acid 66 Ab 8 VL nucleotide 67 Ab 8 VL aminoacid 68 Ab 8 VL CDR 1 amino acid 69 Ab 8 VL CDR 2 amino acid 70 Ab 8 VLCDR 3 amino acid 71 Antibody 10 VH nucleotide 72 Ab 10 VH amino acid 73Ab 10 VH CDR 1 amino acid 74 Ab 10 VH CDR 2 amino acid 75 Ab 10 VH CDR 3amino acid 76 Ab 10 VL nucleotide 77 Ab 10 VL amino acid 78 Ab 10 VL CDR1 amino acid 79 Ab 10 VL CDR 2 amino acid 80 Ab 10 VL CDR 3 amino acid81 Antibody 14 VH nucleotide 82 Ab 14 VH amino acid 83 Ab 14 VH CDR 1amino acid 84 Ab 14 VH CDR 2 amino acid 85 Ab 14 VH CDR 3 amino acid 86Ab 14 VL nucleotide 87 Ab 14 VL amino acid 88 Ab 14 VL CDR 1 amino acid89 Ab 14 VL CDR 2 amino acid 90 Ab 14 VL CDR 3 amino acid 91 Antibody 16VH nucleotide 92 Ab 16 VH amino acid 93 Ab 16 VH CDR 1 amino acid 94 Ab16 VH CDR 2 amino acid 95 Ab 16 VH CDR 3 amino acid 96 Ab 16 VLnucleotide 97 Ab 16 VL amino acid 98 Ab 16 VL CDR 1 amino acid 99 Ab 16VL CDR 2 amino acid 100 Ab 16 VL CDR 3 amino acid 101 Antibody 17 VHnucleotide 102 Ab 17 VH amino acid 103 Ab 17 VH CDR 1 amino acid 104 Ab17 VH CDR 2 amino acid 105 Ab 17 VH CDR 3 amino acid 106 Ab 17 VLnucleotide 107 Ab 17 VL amino acid 108 Ab 17 VL CDR 1 amino acid 109 Ab17 VL CDR 2 amino acid 110 Ab 17 VL CDR 3 amino acid 111 Antibody 18 VHnucleotide 112 Ab 18 VH amino acid 113 Ab 18 VH CDR 1 amino acid 114 Ab18 VH CDR 2 amino acid 115 Ab 18 VH CDR 3 amino acid 116 Ab 18 VLnucleotide 117 Ab 18 VL amino acid 118 Ab 18 VL CDR 1 amino acid 119 Ab18 VL CDR 2 amino acid 120 Ab 18 VL CDR 3 amino acid 121 Antibody 19 VHnucleotide 122 Ab 19 VH amino acid 123 Ab 19 VH CDR 1 amino acid 124 Ab19 VH CDR 2 amino acid 125 Ab 19 VH CDR 3 amino acid 126 Ab 19 VLnucleotide 127 Ab 19 VL amino acid 128 Ab 19 VL CDR 1 amino acid 129 Ab19 VL CDR 2 amino acid 130 Ab 19 VL CDR 3 amino acid 131 Antibody 21 VHnucleotide 132 Ab 21 VH amino acid 133 Ab 21 VH CDR 1 amino acid 134 Ab21 VH CDR 2 amino acid 135 Ab 21 VH CDR 3 amino acid 136 Ab 21 VLnucleotide 137 Ab 21 VL amino acid 138 Ab 21 VL CDR 1 amino acid 139 Ab21 VL CDR 2 amino acid 140 Ab 21 VL CDR 3 amino acid 141 Antibody 22 VHnucleotide 142 Ab 22 VH amino acid 143 Ab 22 VH CDR 1 amino acid 144 Ab22 VH CDR 2 amino acid 145 Ab 22 VH CDR 3 amino acid 146 Ab 22 VLnucleotide 147 Ab 22 VL amino acid 148 Ab 22 VL CDR 1 amino acid 149 Ab22 VL CDR 2 amino acid 150 Ab 22 VL CDR 3 amino acid 151 Antibody 23 VHnucleotide 152 Ab 23 VH amino acid 153 Ab 23 VH CDR 1 amino acid 154 Ab23 VH CDR 2 amino acid 155 Ab 23 VH CDR 3 amino acid 156 Ab 23 VLnucleotide 157 Ab 23 VL amino acid 158 Ab 23 VL CDR 1 amino acid 159 Ab23 VL CDR 2 amino acid 160 Ab 23 VL CDR 3 amino acid 161 Full lengthhuman IL-6 amino acid 162 HIS FLAG tagged human IL-6 163 Soluble IL-6Ra(human) 164 Transmembrane IL-6Ra (human) 165 Mature human IL-6 aminoacid 166 Human gp130 167 Germlined VH FR1 168 Germlined VH FR2 169Germlined VH FR3 170 Germlined VH FR4 171 Germlined VL FR1 172 GermlinedVL FR1 173 Germlined VL FR1 174 Germlined VL FR1 175 F102E mutant IL-6176 S204E mutant IL-6 177 R207E mutant IL-6 178 F106E mutant IL-6 179Q211A mutant IL-6 180 R58E mutant IL-6 181 E200W mutant IL-6 182 R207Lmutant IL-6 183 primer macIL6_5′NdeI 184 primer macIL6_3′NheI 185 primerhIL6_5′NdeI Sequences of antibodies 7, 10, 17 and 18 are germlined.

REFERENCES

All references cited anywhere in this specification, including thosecited anywhere above, are incorporated herein by reference in theirentirety and for all purposes.

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TABLE 7 HCDR1 HCDR2 Kabat numbering 31 32 33 34 35 50 51 52 52A 53 54 5556 57 58 59 60 61 62 63 64 65 CAN022D10 S N Y M I D L Y Y Y A G D T Y YA D S V K G Antibody 2 T R Antibody 3 V Antibody 4 T Antibody 5 Antibody7 Antibody 8 Antibody 10 Antibody 14 Antibody 16 Antibody 17 Antibody 18Antibody 19 Antibody 21 Antibody 22 Antibody 23 HCDR3 LCDR1 Kabatnumbering 95 96 97 98 99 100 100A 100B 100C 100D 101 102 24 25 26 27 2829 30 CAN022D10 W A D D H Y Y Y I — D V R A S Q G I S Antibody 2 GAntibody 3 H Antibody 4 G A Antibody 5 G A Antibody 7 P A W V L Antibody8 P R H Antibody 10 E E E G R G Antibody 14 N P H I Antibody 16 P P LAntibody 17 P P M Antibody 18 P P W L Antibody 19 P S H L I Antibody 21P S H Antibody 22 N N T Y I Antibody 23 A P W V L LCDR1 LCDR2 LCDR3Kabat numbering 31 32 33 34 50 51 52 53 54 55 56 89 90 91 92 93 94 95 9697 CAN022D10 S W L A K A S T L E S Q Q S Y S T P W T Antibody 2 T AAntibody 3 Antibody 4 A Antibody 5 A Antibody 7 W L G — G S Antibody 8 WL G — G S Antibody 10 W L G — G S Antibody 14 A A H A A Antibody 16 W LG — G S Antibody 17 W L G — G S Antibody 18 W L G — G S Antibody 19 W LG — G S Antibody 21 W L G — G S Antibody 22 A A H A A Antibody 23 W L G— G S

1. An isolated binding member for human IL-6, which binds human IL-6with a KD of not more than 30 pM as defined by surface plasmonresonance. 2.-73. (canceled)